Treatment and diagnosis of melanoma

ABSTRACT

The present invention discloses novel agents and methods for diagnosis and treatment of melanoma. Also disclosed are related arrays, kits, and screening methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/228,643, filed Aug. 4, 2016, which is a Continuation of U.S. patentapplication Ser. No. 14/486,477, filed Sep. 15, 2014, now U.S. Pat. No.9,526,710, which is a Continuation of International Applciation No.PCT/US2013/54690 filed Aug. 13, 2013, which claims priority to U.S.Provisional Application No. 61/682,339 filed Aug. 13, 2012 and U.S.Provisional Application No. 61/784,057 filed Mar. 14, 2013. The contentsof the applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to diagnosis and treatment of migrating cancersand melanoma.

BACKGROUND OF THE INVENTION

Melanoma, a malignant tumor, develops from abnormal melanocytes in thelower epidermis and can metastasize to distant sites in the body via theblood and lymph systems. Although it accounts for less than 5% of skincancer cases, melanoma is much more dangerous and responsible for alarge majority of the deaths associated with skin cancer. Across theworld the incidence of melanoma has been increasing at an alarming rate,with a lifetime risk of developing melanoma as high as 1/58 for males inthe U.S. (Jemal et al., 2008, CA: Cancer J. Clin. 58:71-96). Themortality rate of malignant melanoma also continues to rise dramaticallythroughout the world. According to a 2006 WHO report, about 48,000melanoma related deaths occur worldwide per year (Lucas et al. (2006)Environmental Burden of Disease Series. 13. World Health Organization.ISBN 92-4-159440-3). In the United States, it was estimated that almost70,000 people were diagnosed with melanoma during 2010 and approximately9,000 people would be expected to die from the disease (American CancerSociety; www.cancer.org).

Although some conventional cancer therapies have been used in treatingmetastatic melanoma, they are not effective. Metastatic melanomatherefore remains one of the most difficult cancers to treat and one ofthe most feared neoplasms. Accordingly, there is a need for new agentsand methods for diagnosis and treatment of melanoma.

SUMMARY OF INVENTION

This invention addresses the above-mentioned need by providing agentsand methods for diagnosis and treatment of melanoma. The invention isbased, at least in part, on an unexpected discovery of a cooperativemiRNA-protein network deregulated in metastatic melanoma. This networkincludes a number of metastasis suppressor factors and metastasispromoter factors.

In one aspect, the invention features a method for treating cancer,including administering to a subject in need thereof, a LXR agonist,wherein the LXR agonist is administered in an amount sufficient toincrease the expression level or activity level of ApoE to a levelsufficient to slow the spread of metastasis of the cancer.

In another aspect, the invention features a method for treating cancer,including administering to a subject in need thereof, an ApoEpolypeptide in an amount sufficient to treat the cancer.

In another aspect, the invention features a method of slowing the spreadof a migrating cancer, comprising administering to a subject in needthereof, a LXR agonist or an ApoE polypeptide.

In some embodiments of any of the aforementioned methods, the LXRagonist is a LXRβ agonist. In certain embodiments, the LXR agonistincreases the expression level of ApoE at least 2.5-fold in vitro. Incertain embodiments, the LXRβ agonist is selective for LXRβ over LXRα.In other embodiments, the LXRβ agonist has activity for LXRβ that is atleast 2.5-fold greater than the activity of said agonist for LXRα. Insome embodiments, the LXRβ agonist has activity for LXRβ that is atleast 10-fold greater than the activity of said agonist for LXRα. Infurther embodiments, the LXRβ agonist has activity for LXRβ that is atleast 100-fold greater than the activity of said agonist for LXRα. Incertain embodiments, the LXR agonist has activity for LXRβ that is atleast within 2.5-fold of the activity of said agonist for LXRα.

In some embodiments the migrating cancer is metastatic cancer. Themetastatic cancer can include cells exhibiting migration and/or invasionof migrating cells and/or include cells exhibiting endothelialrecruitment and/or angiogenesis. In other embodiments, the migratingcancer is a cell migration cancer. In still other embodiments, the cellmigration cancer is a non-metastatic cell migration cancer.

The migrating cancer can be a cancer spread via seeding the surface ofthe peritoneal, pleural, pericardial, or subarachnoid spaces.Alternatively, the migrating cancer can be a cancer spread via thelymphatic system, or a cancer spread hematogenously.

In particular embodiments, the migrating cancer is a cell migrationcancer that is a non-metastatic cell migration cancer, such as ovariancancer, mesothelioma, or primary lung cancer.

In a related aspect, the invention provides a method for inhibiting orreducing metastasis of cancer comprising administering a LXR agonist oran ApoE polypeptide.

In another aspect, the invention provides a method for inhibitingproliferation or growth of cancer stem cells or cancer initiating cells,including contacting the cell with a LXR agonist or an ApoE polypeptidein an amount sufficient to inhibit proliferation or growth of said cell.

In yet another aspect, the invention provides a method of reducing therate of tumor seeding of a cancer including administering to a subjectin need thereof a LXR agonist or an ApoE polypeptide in an amountsufficient to reduce tumor seeding.

In still a further aspect, the invention provides a method of reducingor treating metastatic nodule-forming of cancer including administeringto a subject in need thereof a LXR agonist or an ApoE polypeptide in anamount sufficient to treat said metastatic nodule-forming of cancer.

In other embodiments, the cancer is breast cancer, colon cancer, renalcell cancer, non-small cell lung cancer, hepatocellular carcinoma,gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer,prostate cancer, sarcoma, or melanoma. In some embodiments, the canceris melanoma. In other embodiments, the cancer is breast cancer. Incertain embodiments, the cancer is renal cell cancer. In furtherembodiments, the cancer is pancreatic cancer. In other embodiments, thecancer is non-small cell lung cancer. In some embodiments the cancer iscolon cancer. In further embodiments, the cancer is ovarian cancer.

In other embodiments, the cancer is a drug resistant cancer. In furtherembodiments, the cancer is resistant to vemurafenib, dacarbazine, aCTLA4 inhibitor, a PD1 inhibitor, or a PDL1 inhibitor.

In some embodiments, the method comprises administering an LXR agonistselected from the list consisting of a compound of any one of FormulaI-IV or any of compound numbers 1-39, or pharmaceutically acceptablesalts thereof. In some embodiments, the LXR agonist is compound 1 or apharmaceutically acceptable salt thereof. In other embodiments, the LXRagonist is compound 2 or a pharmaceutically acceptable salt thereof. Incertain embodiments, the LXR agonist is compound 3 or a pharmaceuticallyacceptable salt thereof. In further embodiments, the LXR agonist iscompound 12 or a pharmaceutically acceptable salt thereof. In someembodiments, the LXR agonist is compound 25 or a pharmaceuticallyacceptable salt thereof. In other embodiments, the LXR agonist iscompound 38 or a pharmaceutically acceptable salt thereof. In furtherembodiments, the LXR agonist is compound 39 or a pharmaceuticallyacceptable salt thereof.

The method can further include administering an antiproliferative,wherein said LXR agonist and said antiproliferative are administered inan amount that together, is sufficient to slow the progression ofmigrating cancer. For example, the antiproliferative and LXR agonist canbe administered within 28 days of each (e.g., within 21, 14, 10, 7, 5,4, 3, 2, or 1 days) or within 24 hours (e.g., 12, 6, 3, 2, or 1 hours;or concomitantly) other in amounts that together are effective to treatthe subject.

In some embodiments, the method comprises administering an ApoEpolypeptide. The ApoE polypeptide fragment can increase the activitylevel or expression level of LRP1 or LRP8, and/or the ApoE polypeptidecan bind to LRP1 or LRP8, the ApoE polypeptide can be the receptorbinding region (RBR) of ApoE. The method can further includeadministering an antiproliferative, wherein said ApoE polypeptide andsaid antiproliferative are administered in an amount that together, issufficient to slow the progression of migrating cancer. For example, theantiproliferative and ApoE polypeptide can be administered within 28days of each (e.g., within 21, 14, 10, 7, 5, 4, 3, 2, or 1 days) orwithin 24 hours (e.g., 12, 6, 3, 2, or 1 hours; or concomitantly) otherin amounts that together are effective to treat the subject.

In some embodiments, the pharmaceutical composition may further comprisean additional compound having antiproliferative activity. The additionalcompound having antiproliferative activity can be selected from thegroup of compounds such as chemotherapeutic and cytotoxic agents,differentiation-inducing agents (e.g. retinoic acid, vitamin D,cytokines), hormonal agents, immunological agents and anti-angiogenicagents. Chemotherapeutic and cytotoxic agents include, but are notlimited to, alkylating agents, cytotoxic antibiotics, antimetabolites,vinca alkaloids, etoposides, and others (e.g., paclitaxel, taxol,docetaxel, taxotere, cis-platinum). A list of additional compoundshaving antiproliferative activity can be found in L. Brunton, B. Chabnerand B. Knollman (eds). Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Twelfth Edition, 2011, McGraw Hill Companies, New York,N.Y.

The method may further include administering a antiproliferativecompound selected from the group consisting of alkylating agents,platinum agents, antimetabolites, topoisomerase inhibitors, antitumorantibiotics, antimitotic agents, aromatase inhibitors, thymidylatesynthase inhibitors, DNA antagonists, farnesyltransferase inhibitors,pump inhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF alphaagonists/antagonists, endothelin A receptor antagonist, retinoic acidreceptor agonists, immuno-modulators, hormonal and antihormonal agents,photodynamic agents, tyrosine kinase inhibitors, antisense compounds,corticosteroids, HSP90 inhibitors, proteosome inhibitors (for example,NPI-0052), CD40 inhibitors, anti-CSI antibodies, FGFR3 inhibitors, VEGFinhibitors, MEK inhibitors, cyclin D1 inhibitors, NF-kB inhibitors,anthracyclines, histone deacetylases, kinesin inhibitors, phosphataseinhibitors, COX2 inhibitors, mTOR inhibitors, calcineurin antagonists,IMiDs, or other agents used to treat proliferative diseases. Examples ofsuch compounds are provided in Tables 1.

In another aspect, the invention features a method for treating melanoma(e.g., metastatic melanoma) in a subject in need thereof. The methodincludes (a) increasing in the subject the expression level or activitylevel of a metastasis suppressor factor selected from the groupconsisting of DNAJA4, Apolipoprotein E (ApoE), LRP1, LRP8, Liver XReceptor (LXR, e.g., both LXR-alpha and LXR-beta), and miR-7 or (b)decreasing in the subject the expression level or activity level of ametastasis promoter factor selected from the group consisting ofmiR-199a-3p, miR-199a-5p, miR-1908, and CTGF.

In the method, the increasing step can be carried out by administeringto the subject one or more of the followings: (i) a polypeptide having asequence of DNAJA4, ApoE or an ApoE fragment, LRP1, LRP8, or LXR; (ii) anucleic acid having a sequence encoding DNAJA4, ApoE, LRP1, LRP8, orLXR; (iii) a ligand for LRP1, LRP8, or LXR; and (iv) an RNAi agentencoding miR-7. Examples of the LRP1 or LRP8 ligand include the receptorbinding portion of ApoE, anti-LRP1 or anti-LRP8 antibodies, and smallmolecule ligands. In one example, increasing the ApoE expression levelcan be carried out by increasing the activity level or expression levelof LXR. Increasing the DNAJA4 expression level can also be carried outby increasing the activity level or expression level of LXR. The LXRactivity level can be increased by administering to the subject a ligandof LXR, such as compounds of Formula I-IV as disclosed below. Theincreasing step can also be carried out by decreasing the expressionlevel or activity level of a microRNA selected from the group consistingof miR-199a-3p, miR-199a-5p, and miR-1908. To this end, one can use anumber of techniques known in the art, including, but not limited to,the miR-Zip technology, Locked Nucleic Acid (LNA), and antagomirtechnology as described in the examples below.

In a another aspect, the invention provides a method for determiningwhether a subject has, or is at risk of having, metastatic melanoma. Themethod includes obtaining from the subject a sample; measuring in thesample (i) a first expression level of a metastasis promoter factorselected from the group consisting of miR-199a-3p, miR-199a-5p,miR-1908, and CTGF, or (ii) a second expression level of a metastasissuppressor factor selected from the group consisting of DNAJA4, ApoE,LRP1, LRP8, LXR, and miR-7; and comparing the first expression levelwith a first predetermined reference value, or the second expressionlevel with a second predetermined reference value. The subject isdetermined to have, or to be at risk of having, metastatic melanoma if(a) the first expression level is above a first predetermined referencevalue or (b) the second expression level is below a second predeterminedreference value. The first and second predetermined reference values canbe obtained from a control subject that does not have metastaticmelanoma. In one embodiment, the measuring step includes measuring boththe first expression level and the second expression level. The samplecan be a body fluid sample, a tumor sample, a nevus sample, or a humanskin sample.

In a another aspect, the invention provides an array having a supporthaving a plurality of unique locations, and any combination of (i) atleast one nucleic acid having a sequence that is complementary to anucleic acid encoding a metastasis promoter factor selected from thegroup consisting of miR-199a-3p, miR-199a-5p, miR-1908, and CTGF or acomplement thereof, or (ii) at least one nucleic acid having a sequencethat is complementary to a nucleic acid encoding a metastasis suppressorfactor selected from the group consisting of DNAJA4, ApoE, LRP1, LRP8,LXR, and miR-7 or a complement thereof. Preferably, each nucleic acid isimmobilized to a unique location of the support. This array can be usedfor metastatic melanoma diagnosis and prognosis.

Accordingly, the invention also provides a kit for diagnosing ametastatic potential of melanoma in a subject. The kit includes a firstreagent that specifically binds to an expression product of a metastasissuppressor gene selected from the group consisting of DNAJA4, ApoE,LRP1, LRP8, LXR, and miR-7; or a second reagent that specifically bindsto an expression product of a metastasis promoter gene selected from thegroup consisting of miR-199a-3p, miR-199a-5p, miR-1908, and CTGF. Thesecond agent can be a probe having a sequence complementary to thesuppressor or promoter gene or a complement thereof. The kit can furthercontain reagents for performing an immunoassay, a hybridization assay,or a PCR assay. In one embodiment, the kit contained the above-mentionedarray.

In a another aspect, the invention provides a method of identifying acompound useful for treating melanoma or for inhibiting endothelialrecruitment, cell invasion, or metastatic angiogenesis. The methodincludes (i) obtaining a test cell expressing a reporter gene encoded bya nucleic acid operatively liked to a promoter of a marker gene selectedfrom the group consisting of miR-199a-3p, miR-199a-5p, miR-1908, andCTGF; (ii) exposing the test cell to a test compound; (iii) measuringthe expression level of the reporter gene in the test cell; (iv)comparing the expression level with a control level; and (v) selectingthe test compound as a candidate useful for treating melanoma or forinhibiting endothelial recruitment, cancer cell invasion, or metastaticangiogenesis, if the comparison indicates that the expression level islower than the control level.

The invention provides another method of identifying a compound usefulfor treating melanoma or for inhibiting endothelial recruitment, cellinvasion, or metastatic angiogenesis. The method includes (i) obtaininga test cell expressing a reporter gene encoded by a nucleic acidoperatively liked to a promoter of a marker gene selected from the groupconsisting of DNAJA4, ApoE, LRP1, LRP8, LXR, and miR-7; (ii) exposingthe test cell to a test compound; (iii) measuring the expression levelof the reporter gene in the test cell; (iv) comparing the expressionlevel with a control level; and (v) selecting the test compound as acandidate useful for treating melanoma or for inhibiting endothelialrecruitment, cancer cell invasion, or metastatic angiogenesis, if thecomparison indicates that the expression level is higher than thecontrol level.

In the above-mentioned identification methods, the reporter gene can bea standard reporter gene (such as LaxZ, GFP, or luciferase gene, or thelike), known in the art, or one of the aforementioned metastasissuppressor genes or metastasis promoter genes. In the methods, thecontrol level can be obtained from a control cell that is the same asthe test cell except that the control cell has not be exposed to thetest compound.

In a another aspect, the invention provides a method for inhibitingendothelial recruitment, inhibiting tumor cell invasion, or treatingmetastatic cancer in a subject in need thereof, by administering to thesubject an agent that inhibits expression or activity of CTGF. Thesubject can be one having a disorder characterized by pathologicalangiogenesis, including but not limited to cancer (e.g., metastaticmelanoma), an eye disorder, and an inflammatory disorder. An example ofthe tumor cell is a metastatic melanoma cell. Examples of the agentinclude an antibody, a nucleic acid, a polypeptide, and a small moleculecompound. In a preferred embodiment, the antibody is a monoclonalantibody.

In a another aspect, the invention provides a method for inhibitingendothelial recruitment, inhibiting tumor cell invasion, or treatingmetastatic cancer in a subject in need thereof, by administering to thesubject an agent that increases expression or activity of miR-7. Anexample of the tumor cell is a metastatic melanoma cell. Examples of theagent include an antibody, a nucleic acid, a polypeptide, and a smallmolecule compound. In one example, the agent has miR-7 activity. Thenucleic acid can be an oligonucleotide. And, the oligonucleotide caninclude a sequence selected from the group consisting of SEQ ID Nos.36-38.

As used herein, “migrating cancer” refers to a cancer in which thecancer cells forming the tumor migrate and subsequently grow asmalignant implants at a site other than the site of the original tumor.The cancer cells migrate via seeding the surface of the peritoneal,pleural, pericardial, or subarachnoid spaces to spread into the bodycavities; via invasion of the lymphatic system through invasion oflymphatic cells and transport to regional and distant lymph nodes andthen to other parts of the body; via haematogenous spread throughinvasion of blood cells; or via invasion of the surrounding tissue.Migrating cancers include metastatic tumors and cell migration cancers,such as ovarian cancer, mesothelioma, and primary lung cancer, each ofwhich is characterized by cellular migration.

As used herein, “slowing the spread of migrating cancer” refers toreducing or stopping the formation of new loci; or reducing, stopping,or reversing the tumor load.

As used herein, “metastatic tumor” refers to a tumor or cancer in whichthe cancer cells forming the tumor have a high potential to or havebegun to, metastasize, or spread from one location to another locationor locations within a subject, via the lymphatic system or viahaematogenous spread, for example, creating secondary tumors within thesubject. Such metastatic behavior may be indicative of malignant tumors.In some cases, metastatic behavior may be associated with an increase incell migration and/or invasion behavior of the tumor cells.

As used herein, “slowing the spread of metastasis” refers to reducing orstopping the formation of new loci; or reducing, stopping, or reversingthe tumor load.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukimias, lymphomas, and the like.

As used herein, “drug resistant cancer” refers to any cancer that isresistant to an antiproliferative in Table 2.

Examples of cancers that can be defined as metastatic include but arenot limited to non-small cell lung cancer, breast cancer, ovariancancer, colorectal cancer, biliary tract cancer, bladder cancer, braincancer including glioblastomas and medullablastomas, cervical cancer,choriocarcinoma, endometrial cancer, esophageal cancer, gastric cancer,hematological neoplasms, multiple myeloma, leukemia, intraepithelialneoplasms, livercancer, lymphomas, neuroblastomas, oral cancer,pancreatic cancer, prostate cancer, sarcoma, skin cancer includingmelanoma, basocellular cancer, squamous cell cancer, testicular cancer,stromal tumors, germ cell tumors, thyroid cancer, and renal cancer.

“Proliferation” as used in this application involves reproduction ormultiplication of similar forms (cells) due to constituting (cellular)elements.

“Cell migration” as used in this application involves the invasion bythe cancer cells into the surrounding tissue and the crossing of thevessel wall to exit the vasculature in distal organs of the cancer cell.

By “cell migration cancers” is meant cancers that migrate by invasion bythe cancer cells into the surrounding tissue and the crossing of thevessel wall to exit the vasculature in distal organs of the cancer cell.

“Non-metastatic cell migration cancer” as used herein refers to cancersthat do not migrate via the lymphatic system or via haematogenousspread.

As used herein, “cell to cell adhesion” refers to adhesion between atleast two cells through an interaction between a selectin molecule and aselectin specific ligand. Cell to cell adhesion includes cell migration.

A “cell adhesion related disorder” is defined herein as any disease ordisorder which results from or is related to cell to cell adhesion ormigration. A cell adhesion disorder also includes any disease ordisorder resulting from inappropriate, aberrant, or abnormal activationof the immune system or the inflammatory system. Such diseases includebut are not limited to, myocardial infarction, bacterial or viralinfection, metastatic conditions, e.g. cancer. The invention furtherfeatures methods for treating a cell adhesion disorder by administeringa LXR agonist or ApoE polypeptide.

As used herein, “cancer stem cells” or “cancer initiating cells” refersto cancer cells that possess characteristics associated with normal stemcells, specifically the ability to give rise to all cell types found ina particular cancer sample. Cancer stem cells are therefore tumorgenicor tumor forming, perhaps in contrast to other non-tumorgenic cancercells. Cancer stem cells may persist in tumors as a distinct populationand cause cancer recurrence and metastasis by giving rise to new tumors.

As used herein, “tumor seeding” refers to the spillage of tumor cellclusters and their subsequent growth as malignant implants at a siteother than the site of the original tumor.

As used herein, “metastatic nodule” refers to an aggregation of tumorcells in the body at a site other than the site of the original tumor.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E and 1F. Systematic Identification of miR-1908,miR-199a-3p, and miR-199a-5p as Endogenous Promoters of Human MelanomaMetastasis (1A) Heat map illustrating variance-normalized microarrayexpression values of miRNAs up-regulated in independent MeWo and A375metastatic derivatives relative to their respective parental cells.Standard deviation changes from the mean of each heat map row areindicated by color map. (1B) miRNAs found to be up-regulated bymicroarray hybridization were validated by qRT-PCR in MeWo-LM2metastatic derivatives. n=3. (1C) Bioluminescence imaging plot of lungmetastatic colonization following intravenous injection of 4×104parental MeWo cells over-expressing the precursors for miR-199a,miR-1908, miR-214, or a control hairpin. Lungs were extracted 63 dayspost-injection and H&E-stained. n=5. (1D) Bioluminescence imaging plotand H&E-stained lungs corresponding to lung metastasis followingintravenous injection of 4×104 LM2 cells expressing a short hairpin(miR-Zip) inhibiting miR-1908 (m1908 KD), miR-199a-3p (m199a3p KD),miR-199a-5p (m199a5p KD), or a control sequence (shCTRL). Lungs wereextracted and H&E-stained 49 days post-injection n=5-8. (1E) Lungcolonization by 2×105 A375-LM3 metastatic derivatives withmiR-Zip-induced silencing of miR-1908, miR-199a-3p, miR-199a-5p, or acontrol sequence was quantified at day 42 by bioluminescence imaging.n=5-8 (1F) The expression levels of miR-199a-3p, miR-199a-5p, andmiR-1908 were determined in a blinded fashion by qRT-PCR in a cohort ofnon-metastatic (n=38) and metastatic (n=33) primary melanoma skinlesions from MSKCC patients. n=71. All data are represented as mean±SEM.*p<0.05, **p<0.01, ***p<0.001. See also FIG. 12.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H. MiR-1908, miR-199a-3p, andmiR-199a-5p Display Dual Cell-Autonomous/Non-Cell-Autonomous Roles inRegulating Melanoma Metastatic Progression (2A) 1×106 parental MeWocells over-expressing miR-199a, miR-1908, or a control hairpin wereinjected subcutaneously into immuno-deficient mice, and primary tumorvolume was monitored over time. n=4-6. (2B) 1×105 parental MeWo cellsover-expressing miR-199a, miR-1908, or a control hairpin were allowed toinvade through a trans-well matrigel-coated insert for 24 hours, and thenumber of cells invaded into the basal side of each insert wasquantified. n=7. (2C-2D) 1×105 highly metastatic MeWo-LM2 (2C) andA375-LM3 (2D) cells with miR-Zip-induced inhibition of miR-199a-3p,miR-199a-5p, miR-1908, or a control sequence were subjected to the cellinvasion assay. n=6-8. (2E) 5×104 MeWo cells over-expressing miR-199a,miR-1908, or a control hairpin were seeded on the bottom of a well, and1×105 human umbilical vein endothelial cells (HUVEC's) were allowed tomigrate towards the cancer cells for 16 hours through a trans-wellinsert. Endothelial recruitment capacity was measured by quantifying thenumber of HUVEC's migrated to the basal side of each insert. n=7.(2F-2G) Endothelial recruitment by 5×104 MeWo-LM2 (2F) and A375-LM3 (2G)cells inhibited for miR-199a-3p, miR-199a-5p, miR-1908, or a controlsequence. n=6-10. (2H) Cumulative fraction plot of the percentage bloodvessel density distribution for metastatic nodules formed followingintravenous injection of 2×105 highly metastatic MeWo-LM2 cells depletedfor miR-199-3p, miR-199a-5p, miR-1908, or a control sequence. Lungsections were immunohistochemically double-stained for human vimentin(blue) and MECA-32 (red), and the percentage MECA-32 positive areawithin each metastatic nodule, demarcated based on vimentin staining,was quantified. n=211 nodules (control KD); n=60 nodules (m199a3p KD);n=138 nodules (m199a5p KD); n=39 nodules (m1908 KD). All data arerepresented as mean±SEM. Scale bar, 100 μm. See also FIG. 13.

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I. Identification of ApoE andDNAJA4 as Common Target Genes of miR-199a and miR-1908 (3A) Heat mapdepicting mRNA levels of ApoE and DNAJA4, measured by qRT-PCR, in poorlymetastatic MeWo cells over-expressing miR-199a, miR-1908, or a controlhairpin and in highly metastatic MeWo-LM2 cells. Color map illustratesstandard deviation changes from the mean of each heat map column. (3B)Heterologous luciferase reporter assays measuring the stability ofwild-type ApoE and DNAJA4 3′UTR/CDS luciferase fusions or miRNAtarget-site mutant ApoE and DNAJA4 3′UTR/CDS fusions in parental MeWocells over-expressing miR-199a, miR-1908, or a control hairpin. n=3-4.(3C) Stability of wild-type ApoE and DNAJA4 3′UTR/CDS luciferase fusionsin MeWo-LM2 cells with silenced expression of miR-199a-3p, miR-199a-5p,miR-1908, or a control sequence. n=4. (3D) Schematic of experimentallyderived model of ApoE and DNAJA4 3′UTR/CDS targeting by miR-199a-3p,miR-199a-5p, and miR-1908. (3E) Luciferase activity of wild-type andmiRNA target-site mutant ApoE and DNAJA4 3′UTR/CDS luciferase fusions inhighly metastatic MeWo-LM2 derivatives and their poorly metastaticparental cell line. n=4. (3F) Matrigel invasion capacity by 1×105MeWo-LM2 cells expressing a control vector or over-expressing ApoE orDNAJA4. n=4. (3G) Endothelial recruitment ability by 5×104 MeWo-LM2cells transduced with a control vector or an over-expression vector forApoE or DNAJA4. n=6. (3H-3I) Poorly metastatic parental MeWo cellstransduced with lentiviral short hairpins targeting ApoE, DNAJA4, or acontrol sequence were assessed for their matrigel invasion capacity (3H)and ability to recruit endothelial cells (3I). n=6-8. All data arerepresented as mean±SEM. Scale bar, 100 μm. See also FIG. 14.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4J and 4K. Direct Targeting ofApoE and DNAJA4 by miR-199a and miR-1908 Promotes Metastatic Invasion,Endothelial Recruitment, and Colonization (4A-4D) Highly metastatic LM2cells expressing a control shRNA or shRNAs targeting ApoE or DNAJA4 inthe context of miR-1908 inhibition (m1908 KD; 4A, 4B) or miR-199a-5pinhibition (m199a5p KD; 4C, 4D) were subjected to the cell invasion (4A,4C) and endothelial recruitment assays (4B, 4D). n=6-8. (4E-4F)Bioluminescence imaging plot and H&E-stained lungs representative oflung metastasis after intravenous injection of 1×105 LM2 cellsexpressing a control hairpin or hairpins targeting ApoE, DNAJA4, or acontrol sequence in the setting of miR-1908 silencing (4E) ormiR-199a-5p silencing (4F). n=5. (4G-4H) Parental MeWo cellsover-expressing ApoE or DNAJA4 or expressing a control vector in thecontext of miR-1908 over-expression were analyzed for the matrigelinvasion (4G) and endothelial recruitment (4H) phenotypes. (4I-4J)A375-LM3 derivatives expressing a control shRNA or shRNAs targeting ApoEand DNAJA4 were transduced with a cocktail of LNAs targetingmiR-199a-3p, miR-199a-5p, and miR-1908 or a control LNA and analyzed inthe matrigel invasion (4I) and endothelial recruitment (4J) assays. n=4.(4K) Blood vessel density distribution, represented in a cumulativefraction plot, for metastatic nodules formed by MeWo-LM2 cells inhibitedfor miR-1908 and transduced with shRNAs targeting ApoE, DNAJA4, or acontrol sequence. Lung sections from FIG. 4E were immunocytochemicallydouble-stained for human vimentin (blue) and the endothelial markerMECA-32 (red). The percentage MECA-32 positive area within eachvimentin-positive nodule was quantified. n=39 nodules (shCTRL); n=97(shAPOE1); n=38 (shAPOE2); n=200 (shDNAJA41); n=19 (shDNAJA42). All dataare represented as mean±SEM. Scale bar, 100 μm. See also FIG. 15.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, 5L and 5M.Melanoma-Cell Secreted ApoE Inhibits Melanoma Invasion and EndothelialRecruitment, while Genetic Deletion of ApoE Accelerates Metastasis(5A-5B) Extracellular ApoE levels quantified by ELISA in conditionedmedia from MeWo-LM2 metastatic derivatives and their parental cells (5A)and LM2 cells silenced for miR-199a-5p, miR-1908, or a control sequence(5B). n=3. (5C) ApoE-neutralizing antibody 1D7 (10-40 μg/mL) or IgG (40μg/mL) was added to the cell media, and matrigel invasion by parentalMeWo cells was assessed. n=4-6. (5D) Endothelial recruitment by parentalMeWo cells in the presence of 1D7 (40 μg/mL) or a control IgG antibody(40 μg/mL). n=4. (5E) The matrigel invasion and endothelial recruitmentphenotypes were assessed in LM2 cells in the presence of bovine serumalbumin (BSA) (100 μM) or recombinant ApoE3 (100 μM) added to the cellmedia. n=7-10. (5F-5G) LM2 cells with silenced expression ofmiR-199a-3p, miR-199a-5p, miR-1908, or a control sequence were examinedfor matrigel invasion capacity (5F) and endothelial recruitment ability(5G) in the presence of IgG or ApoE-neutralizing 1D7 antibodies (40μg/mL). n=5-6. (5H) ApoE levels quantified by ELISA in conditioned mediafrom parental MeWo cells transduced with shRNAs targeting DNAJA4 or acontrol sequence. n=3. (5I-5J) Parental MeWo cells with shRNA-inducedsilencing of DNAJA4 were analyzed for the matrigel invasion (5I) andendothelial recruitment (5J) phenotypes in the presence of either BSA(100 μM) or recombinant ApoE3 (100 μM). n=4. (5K) Array-based ApoEexpression levels in nevi (n=9), primary melanomas (n=6), and distantmelanoma metastases samples (n=19). (5L) Highly metastatic MeWo-LM2cells were incubated in the presence of recombinant ApoE3 or BSA at 100μg/mL. After 24 hours, 4×104 cells were intravenously injected intoNOD-SCID mice, and lung colonization was monitored by bioluminescenceimaging. n=6. (5M) Lung metastasis by 5×104 B16F10 mouse melanoma cellsintravenously injected into ApoE genetically null C57BL/6 mice or theirwild-type control littermates. Lung bioluminescence quantification andrepresentative H&E-stained lungs correspond to 19 days post-injection.n=8-18. All data are represented as mean±SEM. Scale bar, 100 μm.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I and 6J. Identification ofDistinct Melanoma and Endothelial Cell Receptors that Mediate theEffects of ApoE on Melanoma Invasion and Endothelial Recruitment (6A)Matrigel invasion capacity was examined in 1×105 LM2 cells transducedwith siRNAs targeting LDLR, VLDLR, LRP8, LRP1, or a control sequence inthe presence of either BSA (100 μM) or recombinant ApoE3 (100 μM).n=4-7. (6B) 1×105 MeWo-LM2 cells transduced with short hairpinstargeting miR-1908 or a control sequence were transfected with siRNAstargeting LRP1 or a control siRNA and subjected to the matrigel invasionassay. n=4. (6C) Bioluminescence imaging of lung colonization by 1×105LM2 cells transduced with siRNAs targeting LRP1 or a control sequence inthe setting of miR-1908 inhibition. n=5. (6D) 1×105 endothelial cellspre-incubated with BSA (100 μM) or recombinant ApoE3 (100 μM) for 24hours were analyzed for the endothelial recruitment phenotype by 5×105LM2 cells. n=3-4. (6E) 1×105 endothelial cells were transduced withsiRNAs targeting LDLR, VLDLR, LRP1, LRP8, or a control sequence andallowed to migrate in a trans-well system towards LM2 cells inhibitedfor miR-1908 or a control sequence. n=4-12. (6F) Trans-well migration by1×105 endothelial cells in the presence of IgG (40 μg/mL) or 1D7antibodies (40 μg/mL) added to the cell media. n=6-8. (6G) Trans-wellmigration by 1×105 endothelial cells transduced with siRNAs targetingLRP8 or a control sequence in the presence of BSA (100 μM) orrecombinant ApoE3 (100 μM). n=6-7. (6H) 1×105 endothelial cells weretransduced with siRNAs targeting LRP8 or a control sequence, andtrans-well chemotactic migration was assessed along an ApoE gradient.n=6-8. (6I) Endothelial recruitment into matrigel plugs, implantedsubcutaneously above the ventral flank of mice, containing BSA (10μg/mL), VEGF (400 ng/mL)+BSA (10 μg/mL), or VEGF (400 ng/mL)+recombinantApoE3 (10 μg/mL). n=3-6. (6J) Blood vessel density within lungmetastatic nodules formed following intravenous injection of 5×104B16F10 mouse melanoma cells into wild-type or ApoE genetically nullmice. Lung sections from FIG. 5M were immunohistochemically stained forMECA-32, and the percentage MECA-32 positive area within each metastaticnodule, outlined based on cell pigmentation, was quantified. n=17-20.All data are represented as mean±SEM. Scale bar, 100 μm.

FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I AND 7K. Clinical andTherapeutic Cooperativity among miR-199a-3p, miR-199a-5p, and miR-1908in Melanoma Metastasis (7A-7D). Kaplan-Meier curves for the MSKCC cohort(N=71) representing metastasis-free survival of patients as a functionof their primary melanoma lesion's miR-199a-3p (7A), miR-199a-5p (7B),miR-1908 (7C), or aggregate three miRNA expression levels (7D). Patientswhose primary tumors' miRNA expression or aggregate miRNA expressionlevels (sum of the expression values of miR-199a-3p, miR-199a-5p, andmiR-1908) were greater than the median for the population wereclassified as miRNA expression positive (red), while those whose primarytumors expressed the given miRNAs at a level below the median wereclassified as miRNA expression negative (blue). (7E) Lung metastasis byhighly metastatic LM2 cells transfected with LNAs individually targetingeach miR-1908, miR-199a-3p, or miR-199a-5p, a combination of LNAstargeting all three miRNAs, or a control LNA. 48 hourspost-transfection, 1×105 cells were intravenously injected intoimmuno-deficient mice. n=5-6. (7F) Systemic metastasis by 1×105 MeWo-LM2cells transfected with a control LNA (LNA-CTRL) or a cocktail of LNAstargeting miR-1908, miR-199a-3p, miR-199a-5p (LNA-3 miRNAs) 48 hoursprior to intracardiac injection into athymic nude mice. n=5. (7G) Numberof systemic metastatic foci arising from LNA-CTRL and LNA-3 miRNAs LM2cells at day 28 post-intracardiac injection. n=5. (7H-7I)Bioluminescence signal quantification of bone metastasis (7H) and brainmetastasis (7I) at day 28 post-intracardiac injection of LNA-CTRL andLNA-3 miRNAs LM2 cells. n=5. (7J) 4×104 highly metastatic MeWo-LM2 cellswere tail-vein injected into immuno-compromised mice, and the mice wereintravenously treated with a cocktail of in vivo-optimized LNAstargeting miR-1908, miR-199a-3p, and miR-199a-5p at a total dose of 12.5mg/kg or a mock PBS control on a bi-weekly basis for four weeks. Lungcolonization was assessed by bioluminescence imaging, and representativeH&E-stained lungs extracted at day 56 are shown. n=5-6. (7K) Model ofmiRNA-dependent regulation of metastatic invasion, endothelialrecruitment, and colonization in melanoma through targeting ofApoE-mediated melanoma cell LRP1 and endothelial cell LRP8 receptorsignaling.

FIGS. 8A, 8B, 8C, 8D and 8E. MiRNA-dependent targeting of ApoE/LRP1signaling promotes cancer cell invasion and endothelial recruitmentthrough CTGF induction. (8A) A heat-map of variance-normalized CTGFexpression levels, determined by qRT-PCR analysis, in (1) MeWo parentaland MeWo-LM2 cells, (2) MeWo parental cells over-expressing miR-199a,miR-1908, or a control hairpin, and (3) MeWo parental cells transducedwith short hairpins targeting ApoE or a control sequence. Color-mapindicates the standard deviations change from the mean. (8B) CTGF levelsin conditioned media from MeWo parental cells with ApoE knock-downdetermined by ELISA. n=6; p-values based on a one-sided student'st-test. (8C) CTGF levels, quantified by ELISA, in conditioned media fromhighly metastatic MeWo-LM2 cells treated with recombinant ApoE in thesetting of LRP1 knock-down or a control knock-down. n=3-4; p-valuesbased on a one-sided student's t-test. (8D-8E) Parental MeWo cells withshRNA-induced ApoE knock-down were (1) transfected with independentsiRNAs targeting CTGF or a control sequence or (2) incubated in thepresence of a CTGF neutralizing antibody (20 μg/mL) or an IgG controlantibody (20 μg/mL), and the cells were subjected to cell invasion (8D)and endothelial recruitment (8E) assays. n=6-8; p-values based on aone-sided student's t-test; scale bar indicates 100 μM. All data arerepresented as mean±SEM.

FIGS. 9A, 9B and 9C. CTGF mediates miRNA-dependent metastatic invasion,endothelial recruitment, and colonization. (9A) 1×105 parental MeWocells expressing a control hairpin or over-expressing miR-199a ormiR-1908 were subjected to a trans-well cell invasion assay in thepresence of a blocking antibody targeting CTGF (20 μg/mL) or a controlIgG antibody (20 μg/mL) as indicated in the figure. n=4-10; p-valuesbased on a one-sided student's t-test. All data are represented asmean±SEM. (9B) Endothelial recruitment by parental MeWo cells expressinga control hairpin or over-expressing miR-199a or miR-1908. At thebeginning of the assay, a neutralizing antibody targeting CTGF (20μg/mL) or a control IgG antibody (20 μg/mL) were added to endothelialcells as indicated, and 1×105 endothelial cells were allowed to migratetowards 5×104 cancer cells in a trans-well migration assay. n=3-8;p-values based on a one-sided student's t-test. (9C) Bioluminescenceimaging of lung metastasis by 5×104 parental MeWo cells knocked down forCTGF in the setting of miR-199a or miR-1908 over-expression. n=5-6;p-values obtained using a one-way Mann-Whitney t-test. All data arerepresented as mean±SEM.

FIGS. 10A, 10B, 10C, 10D and 10E. Treatment with the LXR agonist GW3965elevates melanoma cell ApoE levels and suppresses cancer cell invasion,endothelial recruitment, and metastatic colonization. (10A-10B) ParentalMeWo cells were incubated in the presence of DMSO or GW3965 at theindicated concentrations. After 48 hours, total RNA was extracted, andthe levels of ApoE (10A) and DNAJA4 (10B) were determined by qRT-PCR.n=3. (10C) Cell invasion by 1×105 parental MeWo cells pre-treated withGW3965 or DMSO for 48 hours. n=6-7. p-values based on a one-sidedstudent's t-test. All data are represented as mean±SEM. (10D)Endothelial recruitment by 5×104 parental MeWo cells pre-treated withGW3965 or DMSO for 48 hours. n=6-7. p-values based on a one-sidedstudent's t-test. (10E) Mice were fed with grain-based chow dietcontaining GW3965 (20 mg/kg) or a control diet. After 10 days, 4×104parental MeWo cells were tail-vein injected into mice, and the mice werecontinuously fed with GW3965-containing chow or a control dietthroughout the experiment. Lung colonization was assessed bybioluminescence imaging. n=5-6; p-values obtained using a one-wayMann-Whitney t-test All data are represented as mean±SEM.

FIGS. 11A and 11B. Identification of miR-7 as an endogenous suppressorof melanoma metastasis. (11A) Bioluminescence imaging plot of lungmetastatic colonization following intravenous injection of 4×104parental MeWo cells expressing a short hairpin (miR-Zip) inhibitingmiR-7 (miR-7 KD). Lungs were extracted 63 days post-injection andH&E-stained. n=5. (11B). Lung metastasis by 4×104 LM2 cellsover-expressing the precursor for miR-7 or a control hairpin. Lungcolonization was monitored weekly by bioluminescence imaging, and lungswere extracted at day 77 post-injection. n=5. All data are representedas mean±SEM; p-values were determined using a one-way Mann-Whitneyt-test. *p<0.05, **p<0.01.

FIGS. 12A, 12B, 12C, 12D, 12E and 12F. In Vivo Selection For HighlyMetastatic Human Melanoma Cell Line Derivatives and Identification ofmiR-199a-3p, miR-199a-5p, and miR-1908 as Metastasis-Promoter miRNAs(12A-12B) Bioluminescence imaging of lung metastasis and representativeimages of H&E-stained lungs corresponding to MeWo-LM2 (12A) and A375-LM3metastatic derivatives (12B) and their respective parental cell lines.4×104 MeWo-Par/MeWo-LM2 cells and 1×105 A375-Par/A375-LM3 cells wereintravenously injected into NOD-SCID mice, and lungs were extracted andH&E stained on day 72 and day 49, respectively. n=4-5. (12C) Expressionlevels of miR-199a-5p, miR-199a-3p, miR-1908, and miR-214 weredetermined by qRT-PCR in A375-LM3 metastatic derivatives and theirparental cells. n=3. (12D) Parental MeWo cells were transduced withretrovirus expressing a control hairpin or a pre-miRNA hairpin constructgiving rise to miR-199a (both miR-199a-3p and miR-199a-5p), miR-1908, ormiR-214. The expression levels of the target miRNAs were determined byqRT-PCR.12 n=3. (12E) H&E-stained lung sections from FIG. 1C wereanalyzed for the number of metastatic nodules resulting from parentalMeWo cells over-expressing miR-199a, miR-1908, or a control hairpin.n=3. (12F) The number of metastatic nodules formed by LM2 cells withsilenced expression of miR-199a-3p, miR-199a-5p, miR-1908, or a controlsequence was analyzed in H&E-stained lung sections from FIG. 1D. n=3.All data are represented as mean±SEM.

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G and 13H. MiR-199a and miR-1908Inhibit Proliferation in vitro and Selectively Promote Cell Invasion andEndothelial Recruitment (13A) 2.5×104 MeWo cells over-expressingmiR-199a, miR-1908, or a control hairpin were seeded in triplicate, andviable cells were counted after 5 days. n=3. (13B) 1×105 poorlymetastatic parental MeWo and highly metastatic LM2 cells were comparedfor their ability to invade though matrigel in a trans-well assay.n=3-4. (13C) 1×105 endothelial cells were seeded in a 6-well plate andallowed to form a monolayer. 2×105 parental MeWo cells over-expressingmiR-199a, miR-1908, or a control hairpin were seeded on top of theendothelial monolayer and incubated for 30 minutes. Each monolayer wassubsequently imaged, and the number of cancer cells adhering toendothelial cells was quantified. n=3. (13D) 1×106 parental MeWo cellsover-expressing miR-199a, miR-1908, or a control hairpin were seeded inlow adherent plates containing cell media supplemented with 0.2%methylcellulose. Following 48 hours in suspension, the numbers of deadand viable cells were quantified. n=3. (13E) 5×105 parental MeWo cellsover-expressing miR-199a, miR-1908, or a control hairpin were seeded ina 6-well plate and incubated in low-serum media for 48 hours, afterwhich the number of viable cells was quantified. n=4. (13F) Colonyformation by parental MeWo cells over-expressing miR-199a, miR-1908, ora control hairpin. 50 cells were seeded in a 6-cm plate, and the numberof colonies formed was quantified 2 weeks later. n=4. (13G) 5×104parental MeWo and LM2 cells were seeded on the bottom of a well andassessed for their ability to recruit endothelial cells. n=6-8. (13H)Percentage blood vessel density, shown as a cumulative fraction plot,for metastatic nodules formed by parental MeWo cells over-expressingmiR-199a, miR-1908, or a control hairpin. Lung sections from FIG. 1Cwere immunohistochemically double-stained for human vimentin andMECA-32, and the MECA-32 positive area relative to the total nodulearea, given by human vimentin staining, was quantified using ImageJ.n=43 nodules (control); n=117 nodules (miR-199a OE); n=55 nodules(miR-1908 OE). All data are represented as mean±SEM. Scale bar, 100 μm.

FIG. 14A, 14B, 14C, 14D, 14E, 14F, 14G. MiR-199a and miR-1908Convergently and Cooperatively Target ApoE and DNAJA4 (14A) Venn diagramshowing the integrative experimental approach that lead to theidentification of putative target genes common to miR-199a-3p,miR-199a-5p, and miR-1908. Transcriptomic profiling of genesdown-regulated by greater than 1.5-fold upon each miRNA over-expressionwere overlapped with genes up-regulated by more than 1.5-fold upon eachmiRNA silencing and with genes down-regulated by more than 1.5-fold inmetastatic LM2 cells relative to their parental cell line. (14B, 14C,14D) Expression levels of ApoE and DNAJA4 measured by qRT-PCR inparental MeWo cells over-expressing miR-199a, miR-1908, or a controlhairpin (14B), in parental MeWo cells and their highly metastatic LM2derivative cell line (14C), and in MeWo-LM2 cells with miR-Zip-basedsilencing of miR-199a-3p, miR-199a-5p, miR-1908, or a control sequence(14D). n=3. (14E) Heterologous luciferase reporter assays measuring thestability of miR-199a-3p, miR-199a-5p, or miR-1908 target site mutantApoE and DNAJA4 3′UTR/CDS luciferase fusions in highly metastatic LM2cells with inhibition of miR-199a-3p, miR-199a-5p, miR-1908, or acontrol sequence. n=3-4. (14F) MeWo-LM2 cells were transduced withretrovirus expressing a control vector or an over-expression vectorgiving rise to ApoE or DNAJA4. The expression levels of the target geneswere determined by qRT-PCR. (14G) Expression levels of ApoE and DNAJA4,determined by qRT-PCR, in parental MeWo cells were transduced withlentiviral shRNAs targeting ApoE, DNAJA4, or a control sequence. Alldata are represented as mean±SEM.

FIGS. 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, 15I and 15K. EpistaticInteractions between miR-199a/miR-1908 and ApoE/DNAJA4 (15A, 15B, 15Cand 15D). MeWo-LM2 cells were transduced with lentiviral shRNAstargeting ApoE (15A, 15C), DNAJA4 (15B, 15D), or a control shRNA in thesetting of miR-Zip-induced silencing of miR-1908 (15A, 15B), miR-199a-5p(15C, 15D), or a control sequence. The levels of the target genes wereanalyzed by qRT-PCR. (15E) Bioluminescence imaging of lung metastasis by1×105 LM2 cells expressing a control hairpin or shRNAs (independent fromthe shRNAs used in FIG. 4E) targeting ApoE, DNAJA4, or a controlsequence in the setting of miR-1908 inhibition. Representativebioluminescence images and H&E-stained lungs correspond to day 42post-injection. n=5. (15F-15G) The expression levels of ApoE and DNAJA4were analyzed by qRT-PCR in parental MeWo cells transduced withretrovirus expressing a control vector or an over-expression vector forApoE or DNAJA4 in the setting of miR-1908 (15F) or miR-199a (15G)over-expression. (15H-15I). Parental MeWo cells over-expressing ApoE orDNAJA4 or expressing a control vector in the setting of miR-199aover-expression were examined for the invasion (15H) and endothelialrecruitment (15I) phenotypes. n=7-8. (15J) Bioluminescence imaging oflung metastasis by 4×104 parental MeWo cells over-expressing ApoE orDNAJA4 or expressing a control vector in the setting of miR-1908over-expression. Representative bioluminescence images and H&E-stainedlungs correspond to day 56 post-injection n=4-8. (15K). Expressionlevels of ApoE and DNAJA4, determined by qRT-PCR, in highly metastaticA375-LM3 derivatives transduced with lentivirus expressing shRNAconstructs targeting ApoE and DNAJA4 or a control sequence. All data arerepresented as mean±SEM. Scale bar, 100 μm.

FIGS. 16A, 16B, 16C, 16C, 16D, 16E, 16F, 16G, 16H and 16I. ExtracellularApoE Inhibits Melanoma Invasion and Endothelial Recruitment PhenotypesIndependent of Any Effects on Cancer or Endothelial Cell Proliferationand Survival (16A) Extracellular ApoE levels were measured by ELISA inconditioned media from MeWo cells over-expressing miR-199a, miR-1908, ora control hairpin. n=3. (16B-16C) 3×104 MeWo-LM2 cells (16B) orendothelial cells (16C) were cultured in the presence of BSA (100 μM) orAPOE (100 μM), and cell proliferation was monitored over time bycounting the number of viable cells at each indicated time-point. n=3.(16D-16E) Survival of MeWo-LM2 cells (16D) or endothelial cells (16E) inthe context of serum starvation in the presence of BSA (100 μM) or APOE(100 μM). n=3. (16F-16G) The mRNA expression levels of ApoE wereassessed in parental MeWo cells transduced with lentivirus expressing acontrol hairpin or short hairpin constructs targeting DNAJA4 (16F) andin LM2 cells transduced with retrovirus expressing a control vector oran over-expression vector for DNAJA4 (16G). n=3. (H-I) LM2 cellstransduced with retrovirus expressing a control vector or anover-expression vector for DNAJA4 were assessed for their ability toinvade through matrigel (16H; n=6-8) and recruit endothelial cells in atrans-well assay (16I; n=4) in the presence of IgG (40 m/mL) or 1D7 (40m/mL) ApoE neutralization antibodies. All data are represented asmean±SEM.

FIGS. 17A, 17B, 17C, 17D and 17E. ApoE Inhibits Cell Invasion andEndothelial Recruitment by Targeting Melanoma Cell LRP1 and EndothelialCell LRP8 Receptors (17A) 1×105 LM2 cells transduced with siRNAs againstLRP1 or a control sequence were analyzed for the ability to invadethrough matrigel. n=9-12. (17B) 1×105 MeWo-LM2 cells inhibited formiR-199a-5p or a control sequence were transfected with siRNAs targetingLRP1 or a control siRNA and examined for their matrigel invasioncapacity. n=4. (17C) Representative H&E-stained lungs extracted at day56 from NOD-SCID mice injected with MeWo-LM2 miR-1908 KD cellstransduced with a control siRNA or siRNAs targeting LRP1 (See FIG. 6C).(17D-17E) 1×105 endothelial cells were transfected with siRNAs targetingLRP8 or a control sequence and allowed to trans-well migrate towards5×104 MeWo-LM2 cells expressing a short control hairpin (17D; n=8) or5×104 MeWo-LM2 cells inhibited for miR-199a-5p or a control sequence(17E; n=4). All data are represented as mean±SEM. Scale bar, 100 μm.

FIGS. 18A, 18B, and 18C. LNA-Based Inhibition of miR-199a and miR-1908Suppresses Melanoma Metastasis (18A) In vitro cell proliferation by2.5×104 MeWo-LM2 cells transduced with a control LNA or a cocktail ofLNAs targeting miR-199a-3p, miR199a-5p and miR-1908. The number ofviable cells was quantified after five days. n=3. (18B) Lungcolonization by highly metastatic A375-LM3 derivatives transfected witha control LNA or a cocktail of LNAs targeting miR-199a-3p, miR199a-5p,and miR-1908. 48 hours post-transfection, 5×105 cells were injectedintravenously into NOD-SCID mice, and lung colonization was determinedby measuring bioluminescence 35 days later. n=5-6. (18C) The weight ofmice treated with a cocktail of LNAs targeting the three miRNAs or amock PBS control treatment (FIG. 7J) was monitored bi-weekly. n=5-6. Alldata are represented as mean±SEM.

FIGS. 19A, 19B, 19C, 19D, 19E, 19F and 19G. Activation of LXRβ SignalingSuppresses Melanoma Cell Invasion and Endothelial Recruitment. (19A)Heat-map depicting microarray-based expression levels of LXR and RXRisoforms in the NCI-60 melanoma cell line collection. The heat map forthese genes is extracted from the larger nuclear hormone receptor familyheat map (FIG. 20). Color-map key indicates the change in standarddeviations for the expression value of each receptor relative to theaverage expression value of all microarray-profiled genes (>39,000transcript variants) in each cell line. (19B) Cell invasion by 1×105MeWo, 5×104 HT-144, 5×105 SK-Mel-2, and 5×104 SK-Mel-334.2 humanmelanoma cells. Cells were treated with DMSO, GW3965, T0901317, orBexarotene at 1 μM for 72 hours and subjected to a trans-well matrigelinvasion assay. n=4-8. (19C) 5×104 MeWo, HT-144, SK-Mel-2, andSK-Mel-334.2 human melanoma cells were tested for their ability torecruit 1×105 endothelial cells in a trans-well migration assay,following treatment of the melanoma cells with DMSO, GW3965, T0901317,or Bexarotene at 1 μM for 72 hours. n=4-8. (19D-19E) 1×105 MeWo (19D)and 1×105 HT-144 (19E) melanoma cells expressing a control shRNA orshRNAs targeting LXRα or LXRβ were subjected to the cell invasion assayfollowing treatment of the cells with DMSO, GW3965, or T0901317 at 1 μMfor 72 hours. n=4-12. (19F-19G) 5×104 MeWo (19F) and 5×104 HT-144 (19G)cells, transduced with lentiviral shRNAs targeting LXRα or LXRβ or acontrol shRNA, were treated with DMSO, GW3965, or T0901317 at 1 μM for72 hours and tested for their ability to recruit 1×105 endothelial cellsin a trans-well migration assay. n=7-8. All data are represented asmean±SEM. Scale bar, 50 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

FIGS. 20A, 20B, 20C, 20D, 20E, 20F and 20G. Analysis of Nuclear HormoneReceptor Expression in Melanoma and Effects of LXR and RXR Agonists onIn Vitro Cell Growth, Related to FIG. 19(A-G). (20A) Heat-map showingmicroarray-based expression levels of all nuclear hormone receptorfamily members across the NCI-60 collection of melanoma lines. Theexpression levels of each receptor is presented as the number ofstandard deviations below or above the average expression levels of allgenes (>39,000 transcript variants) detected by the microarray in eachrespective cell line. (20B) 2.5×104 MeWo, HT-144, or SK-Mel-334.2 humanmelanoma cells were seeded in 6-well plates and cultured in the presenceof DMSO, GW3965, T0901317, or Bexarotene at 1 μM. Viable cells werecounted on day 5 post-seeding. n=3-6. (20C) 2.5×104 MeWo, HT-144, orSK-Mel-334.2 cells were plated in triplicates and incubated in mediacontaining DMSO, GW3965, T0901317, or Bexarotene at 1 μM for 5 days,after which the number of dead cells was quantified using trypan bluedead cell stain. n=3. (20D-20G) Relative expression of LXRα and LXRβ,determined by qRT-PCR, in MeWo (20D, 20E) and HT-144 (20F, 20G) humanmelanoma cells expressing a control shRNA or shRNAs targeting LXRα orLXRβ. All data are represented as mean±SEM.

FIGS. 21A, 21B, 21C, 21D, 21E, 21F, 21G, 21H, 21J, 21K and 21L.Therapeutic LXR Activation Inhibits Melanoma Tumor Growth. (21A-21B)Primary tumor growth by 5×104 B16F10 mouse melanoma cells subcutaneouslyinjected into C57BL/6-WT mice. Following tumor growth to 5-10 mm3 involume, mice were continuously fed a control chow or a chow supplementedwith GW3965 (20 mg/kg/day or 100 mg/kg/day) (21A) or T0901317 (20mg/kg/day) (21B). Representative tumor images shown correspond to tumorsextracted at the final day (d12). n=10-18 (21A), 8-10 (21B). (21C-21E)Primary tumor growth by 1×106 MeWo (21C), 7.5×105 SK-Mel-334.2 (21D),and 2×106 SK-Mel-2 (21E) human melanoma cells subcutaneously injectedinto immunocompromised mice. Following tumor growth to 5-10 mm3 involume, mice were randomly assigned to a control diet or a dietsupplemented with GW3965 (20 mg/kg or 100 mg/kg, as indicated). Tumorimages shown correspond to last day of measurements. n=6-34 (21C), 8(21D), 5 (21E). (21F) 5×104 B16F10 cells were injected subcutaneouslyinto C57BL/6-WT mice. Upon tumor growth to 150 mm3, mice were fedcontinuously with a control chow or a chow containing GW3965 (150mg/kg), and tumor growth was measured daily. n=6-13. (21G-21I) Mouseoverall survival following subcutaneous grafting of 5×104 B16F10 (21G),1×106 MeWo (21H), and 7.5×105 SK-Mel-334.2 cells (21I) into mice thatwere administered a normal chow or a chow supplemented with GW3965 (100mg/kg) upon formation of tumors measuring 5-10 mm3 in volume. n=6-9(21F), 4-7(21H), 3-6 (21I). (21J, 21K, 21L) Tumor endothelial celldensity, determined by immunohistochemical staining for the mouseendothelial cell antigen MECA-32 (21J), tumor cell proliferation,determined by staining for the proliferative marker Ki-67 (21K), andtumor cell apoptosis, determined by staining for cleaved caspase-3(21L), in subcutaneous melanoma tumors formed by 1×106 MeWo humanmelanoma cells in response to mouse treatment with a control diet or aGW3965-supplemented diet (20 mg/kg) for 35 days. n=5. Tumor volume wascalculated as (small diameter)2×(large diameter)/2. All data arerepresented as mean±SEM. Scale bars, 5 mm (21A, 21B, 21C, 21D), 50 μm(21J, 21K), 25 μm (21L).

FIG. 22. LXRβ Agonism Suppresses Melanoma Tumor Growth, Related to FIG.21(A-E). Weight measurements of mice fed a control diet or a dietsupplemented with GW3965 (20 mg/kg/day or 100 mg/kg/day) or T0901317 (20mg/kg) for 65 days. n=5-6.

FIGS. 23A, 23B, 23C, 23D, 23E, 23F, 23G, 23H, 23I, 23J and 23K. LXRAgonism Suppresses Melanoma Metastasis to the Lung and Brain. (23A) MeWocells were pre-treated with DMSO or GW3965 (1 μM) for 48 hours and 4×104cells were intravenously injected via the tail-vein into NOD Scid mice.Lung colonization was monitored by weekly bioluminescence imaging.Representative H&E-stained lungs correspond to the final day (d70) areshown. n=4-5. (23B-23C) Bioluminescence imaging of lung metastasis by4×104 MeWo cells intravenously injected into NOD Scid mice that were feda control chow or a chow containing GW3965 (20 mg/kg) or T0901317 (20mg/kg) starting 10 days prior to cancer cell injection. RepresentativeH&E-stained lungs correspond to final imaging day n=5-6. (23B-23C)Bioluminescence imaging of lung metastasis by 4×104 MeWo cellsintravenously injected into NOD Scid mice that were fed a control chowor a chow containing GW3965 (20 mg/kg) or T0901317 (20 mg/kg) starting10 days prior to cancer cell injection. Representative H&E-stained lungscorrespond to final imaging day n=5-6. (23F) Systemic and brain photonflux following intracardiac injection of 1×105 MeWo brain metastaticderivative cells into athymic nude mice that were fed a control diet ora GW3965-supplemented diet (100 mg/kg) starting on day 0 post-injection.n=7. (23G) Schematic of experimental orthotopic metastasis model used toassess the ability of GW3965 treatment to suppress lung metastasispost-tumor excision. (23H) Ex-vivo lung photon flux, determined bybioluminescence imaging, in NOD Scid mice that were administered acontrol chow or a chow containing GW3965 (100 mg/kg) for 1 monthfollowing the excision of size-matched (˜300-mm3 in volume) subcutaneousmelanoma tumors formed by 1×106 MeWo melanoma cells. Representativelungs stained for human vimentin are also shown. n=7-9. (231) 4×104 MeWocells were intravenously injected into NOD Scid mice. Followinginitiation of metastases, detected by bioluminescence imaging on d42,mice were administered a control diet or a GW3965 diet (100 mg/kg) asindicated, and lung colonization progression was measured weekly. n=6.(23J) Number of macroscopic metastatic nodules in H&E-stained lungsextracted at the final day (d77) from NOD Scid mice administered acontrol diet or a diet supplemented with GW3965 (100 mg/kg), asindicated in (23I). n=4-5. (23K) Overall mouse survival followingintravenous injection of 4×104 MeWo cells into NOD-Scid mice that werecontinuously fed a control chow or a GW3965-supplemented chow (20 mg/kg)starting 10 days prior to cancer cell injection. n=5-6. All data arerepresented as mean±SEM.

FIGS. 24A, 24B, 24C, 24D, 24E and 24F. Suppression of Genetically-DrivenMelanoma Progression by LXR Activation Therapy. (24A) Overall survivalof Tyr::CreER; BrafV600E/+; Ptenlox/+C57BL/6 mice following generalmelanoma induction by intraperitoneal administration of 4-HT (25 mg/kg)on three consecutive days. After the first 4-HT injection, mice wererandomly assigned to a control diet or a diet supplemented with GW3965(100 mg/kg). n=10-11. (24B) Melanoma tumor burden, expressed as thepercentage of dorsal skin area, measured on day 35 in Tyr::CreER;BrafV600E/+; Ptenlox/lox mice administered a control chow or a chowsupplemented with GW3965 (100 mg/kg) upon melanoma induction asdescribed in (24A). n=4-5. (24C) Number of macroscopic metastaticnodules to the salivary gland lymph nodes detected post-mortem inTyr::CreER; BrafV600E/+; Ptenlox/lox mice that were fed a control chowor a chow containing GW3965 (100 mg/kg) following global induction ofmelanoma progression as described in (24A). n=7-8. (24D) Tumor growthfollowing subcutaneous injection of 1×105 BrafV600E/+; Pten−/−;CDKN2A−/− primary melanoma cells into syngeneic C57BL/6-WT mice. Upontumor growth to 5-10 mm3 in volume, mice were fed with a control chow ora chow supplemented with GW3965 (100 mg/kg). n=16-18. (24E) Overallsurvival of C57BL/6-WT mice subcutaneously injected with 1×105BrafV600E/+; Pten−/−; CDKN2A−/− melanoma cells and treated with a GW3965diet (100 mg/kg) or a control diet following tumor growth to 5-10 mm3 involume. n=7-8. (24F) Lung colonization by 1×105 BrafV600E/+; Pten−/−;CDKN2A−/− primary melanoma cells intravenously injected into C57BL/6-WTmice. Immediately following cancer cell injection, mice were randomlyassigned to a control diet or a GW3965-supplemented diet (100 mg/kg) forthe remainder of the experiment. n=14-15. All data are represented asmean±SEM. Scale bar, 2 mm (24B), 5 mm (24D).

FIGS. 25A and 25B. LXR-Mediated Suppression of Melanoma Progression in aGenetically-Driven Melanoma Mouse Model, Related to FIG. 24(A-C). (25A)Overall survival of Tyr::CreER; BrafV600E/+; Ptenlox/lox C57BL/6 micefollowing general melanoma induction by intraperitoneal administrationof 4-HT (25 mg/kg) on three consecutive days. After the first 4-HTinjection, mice were randomly assigned to a control diet or a dietsupplemented with GW3965 (100 mg/kg). n=7. (25B) Representative imagesof Tyr::CreER; BrafV600E/+; Ptenlox/lox C57BL/6 mice fed a control dietof GW3965-supplemented diet (100 mg/kg) taken 43 days following melanomainduction by intraperitoneal 4-HT administration.

FIG. 26. A List of the 50 most upregulated genes in MeWo human melanomacells in response to GW3965 treatment.

FIGS. 27A, 27B, 27C, 27D, 27E, 27F, 27G, 27H, 27I, 27J and 27K. LXRβActivation Induces ApoE Expression in Melanoma Cells; ApoE mediatesLXRβ-Dependent Suppression of In Vitro Melanoma Progression Phenotypes.(27A, 27B, 27C) MeWo (27A), HT-144 (27B), and WM-266-4 (27C) humanmelanoma cells were treated with GW3965 or T0901317 at the indicatedconcentrations for 48 hours, and the expression levels of ApoE wereanalysed by qRT-PCR. n=3. (27D) Extracellular ApoE protein levels,quantified by ELISA, in serum-free conditioned media collected fromHT-144 human melanoma cells treated with DMSO, GW3965, or T0901317 at 1μM for 72 hours. n=3-4. (27E-27F) 5×104 HT-144 cells, treated with DMSO,GW3965, or T0901317 at 1 μM for 72 hours, were tested for the cellinvasion (27E) and endothelial recruitment phenotypes (27F) in thepresence of an ApoE neutralization antibody (1D7) or an IgG controlantibody added at 40 μg/mL to each trans-well at the start of the assay.n=4. (27G-27H) Cell invasion (27G) and endothelial recruitment (27F) by1×105 and 5×104 MeWo cells, respectively, expressing a control shRNA oran shRNA targeting ApoE and treated with DMSO or GW3965 at 1 μM for 72hours prior to each assay. n=7-8. (27I-27J) Relative ApoE expression,quantified by qRT-PCR, in MeWo (I) and HT-144 (27J) cells transducedwith a control shRNA or shRNAs targeting LXRα or LXRβ and subsequentlytreated with DMSO, GW3965, or T0901317 at 1 μM for 48 hours. n=3-9.(27K) Extracellular ApoE protein levels, measured by ELISA, inserum-free conditioned media harvested from HT-144 cells transduced witha control shRNA or an shRNA targeting LXRα or LXRβ and treated with DMSOor GW3965 at 1 μM for 72 hours. n=3. All data are represented asmean±SEM. Scale bar, 50 μm.

FIGS. 28A, 28B, 28C, 28D, 28E, 28F, 28G, 28H and 281. LXRβ ActivationSuppresses Melanoma Invasion and Endothelial Recruitment byTranscriptionally Enhancing Melanoma-Cell ApoE Expression. (28A)Luciferase activity driven off the ApoE promoter fused downstream ofmulti-enhancer element 1 (ME.1) or multi-enhancer element 2 (ME.2)sequences and transfected into MeWo cells treated with DMSO, GW3965, orT0901317 at 1 μM for 24 hours. n=4-8. (28B) Extracellular ApoE proteinlevels were quantified by ELISA in serum-free conditioned mediaharvested from MeWo cells treated with DMSO, GW3965, or T0901317 at 1 μMfor 72 hours. n=3-4. (28C) Cell invasion by 1×105 MeWo cells pre-treatedwith DMSO, GW3965, or T0901317 at 1 μM for 72 hours. At the start of theassay, an ApoE neutralization antibody (1D7) or an IgG control antibodywas added at 40 μg/mL to each trans-well, as indicated. n=7-8. (28D)5×104 MeWo cells, pre-treated with DMSO, GW3965, or T0901317 at 1 μM for72 hours, were tested for their ability to recruit 1×105 endothelialcells in the presence of 1D7 or IgG antibodies at 40 μg/mL. n=6-8. (28E)Extracellular ApoE protein levels, quantified by ELISA, in serum-freeconditioned media from SK-Mel-334.2 primary human melanoma cells treatedwith DMSO or GW3965 at 1 μM for 72 hours. n=4. (28F-28G) 5×104SK-Mel-334.2 cells, pre-treated with GW3965 at 1 μM for 72 hours, weresubjected to the cell invasion (28F) and endothelial recruitment (28G)assays in the presence of 1D7 or IgG antibodies at 40 μg/mL. n=7-8.(28H) Activity of the ApoE promoter fused to ME.1 or ME.2 enhancerelements was determined through measuring luciferase reporter activityin MeWo cells expressing a control shRNA or shRNAs targeting LXRα orLXRβ in the presence of DMSO or GW3965 (1 μM) for 24 hours. n=3-8. (28I)Extracellular ApoE protein levels, quantified by ELISA, were assessed inserum-free conditioned media collected from human MeWo melanoma cellsexpressing a control shRNA or shRNAs targeting LXRα or LXRβ in responseto treatment with GW3965 or T0901317 (1 μM) for 72 hours. n=3-8. Alldata are represented as mean±SEM. Scale bar, 50 μm.

FIGS. 29A, 29B, 29C, 29D, 29E, 29F, 29G, 29H, 29I, 29J and 29K.Therapeutic Delivery of LXR Agonists Upregulates Melanoma-Derived andSystemic ApoE Expression. (29A-29B) ApoE expression levels, quantifiedby qRT-PCR, in subcutaneous tumors formed by B16F10 mouse melanoma cellsinjected into C57BL/6 mice. After 5-mm3 tumor formation, mice were fed acontrol diet or diet containing GW3965 (20 mg/kg) (29A) or T0901317 (20mg/kg) (29B) for 7 days. n=3-4. (29C, 29D, 29E) ApoE transcriptexpression in primary tumors (29C), lung metastases (29D), and brainmetastases (29E) formed by MeWo human melanoma cells grafted onto NODScid mice that were administered control chow or chow supplemented withGW3965 (20 mg/kg). ApoE levels were assessed on day 35 (29C), day 153(29D), and day 34 (29E) post-injection of the cancer cells. n=3-5. (29F)Relative expression levels of LXRα, LXRβ, and ApoE were determined byqRT-PCR in B16F10 mouse melanoma cells expressing a control hairpin oran shRNA targeting mouse LXRα (sh_mLXRα), mouse LXRβ (sh_mLXRβ), ormouse ApoE (sh_mApoE). (29G-29H) ApoE (29G) and ABCA1 (29H) mRNA levels,measured by qRT-PCR, in B16F10 cells expressing a control shRNA orshRNAs targeting mouse LXRβ or mouse ApoE. The cells were treated withDMSO or GW3965 at 5 μM for 48 hours. n=3. (29I) ABCA1 mRNA levels,measured by qRT-PCR, in systemic white blood cells extracted fromLXRα−/− or LXRβ−/− mice fed a control diet or a GW3965-supplemented diet(20 mg/kg) for 10 days. n=3-4. (29J) Relative expression of ApoE mRNA,expressed as the frequency of SAGE tags, in mouse skin and lung tissueswas determined using the public mSAGE Expression Matrix databaseavailable through the NCI-funded Cancer Genome Anatomy Project (CGAP).(29K) Relative expression of ApoE mRNA, determined by qRT-PCR, in MeWomelanoma cells dissociated from lung metastatic nodules (LM2) or primarytumors relative to control unselected MeWo parental cells. n=3.

FIGS. 30A, 30B, 30C, 30D, 30E, 30F, 30G, 30H and 301. LXRβ AgonismSuppresses Melanoma Tumor Growth and Metastasis by InducingMelanoma-Derived and Systemic ApoE Expression. (30A) Western blotmeasurements of ApoE protein levels in adipose, lung, and brain tissuelysates extracted from wild-type mice fed with a control chow or a chowsupplemented with GW3965 (20 mg/kg) or T0901317 (20 mg/kg) for 10 days.(30B) Quantification of ApoE protein expression based on western blotsshown in (30A). Total tubulin was used as an endogenous control fornormalization. n=3-5. (30C) Expression levels of ApoE, determined byqRT-PCR, in systemic white blood cells from mice fed a control diet or adiet supplemented with GW3965 or T0901317 at 20 mg/kg for 10 days.n=3-6. (30D) B16F10 control cells or B16F10 cells expressing shRNAstargeting mouse LXRα (sh_mLXRα) or mouse LXRβ (sh_mLXRβ) weresubcutaneously injected into C57BL/6-WT, LXRα−/−, or LXRβ−/− mice. Oncethe tumors reached 5-10 mm3 in volume, mice were fed a control diet or adiet supplemented with GW3965 (20 mg/kg) for 7 days, after which finaltumor volume was measured. Representative tumor images extracted at theend point are shown in the right panel. n=6-18. (30E) ApoE transcriptlevels, quantified by qRT-PCR, in systemic white blood cells extractedfrom LXRα−/− or LXRβ−/− mice fed a control diet or a GW3965-supplementeddiet (20 mg/kg) for 10 days. n=3-5. (30F) Subcutaneous tumor growth by5×104 B16F10 control cells or B16F10 cells expressing an shRNA targetingmouse ApoE (sh_mApoE) in C57BL/6-WT or ApoE−/− mice. Following theformation of tumors measuring 5-10 mm3 in volume, mice were fed acontrol diet or a diet supplemented with GW3965 (20 mg/kg) for 7 days,and final tumor volume was quantified. Representative images of tumorsextracted at the final day of measurement (d12) are shown on the right.n=8-18. (30G) Lung colonization by 5×104 B16F10 cells transduced with acontrol shRNA or sh_mApoE and intravenously injected into C57BL/6-WT orApoE−/− mice. Starting 10 days prior to cancer cell injection, mice wereassigned to a control diet or a GW3965-supplemented diet (20 mg/kg)treatment. Lung metastasis was quantified on d22 by bioluminescenceimaging. Representative lungs extracted at the end point (d22) are shownin the right panel. n=5-10. (30H) ApoE protein expression, determined byblinded immunohistochemical analysis, in non-metastatic (n=39) andmetastatic (n=34) primary melanoma skin lesion samples obtained frompatients at MSKCC. The fraction of ApoE-positively staining cell areawas quantified as a percentage of total tumor area. (30I) Kaplan-Meiercurves for the MSKCC cohort (n=71) depicting the metastasis-freesurvival of patients as a function of ApoE protein expression inpatients' primary melanoma lesions. Melanomas that had ApoE levels abovethe median of the population were classified as ApoE-positive (pos),whereas tumors with ApoE expression below the median were classified asApoE-negative (neg). All data are represented as mean±SEM. Scale bar, 5mm (30D and 30F), 100 μm (30H).

FIGS. 31A, 31B, 31C, 31D, 31E, 31F, 31G, 31H and 31I. Activation of LXRβSuppresses the In Vivo Growth of Melanoma Lines Resistant to Dacarbazineand Vemurafenib. (31A) In vitro cell growth by 2.5×104 B16F10 parentalcells and in vitro-derived B16F10 DTIC-resistant cells in response tovarying doses of dacarbazine (DTIC) added to the cell media for 4 days.n=3. (31B-31D) Tumor growth by 5×104 DTIC-sensitive B16F10 parentalcells (31B) or 5×104 DTIC-resistant B16F10 cells (31C) subcutaneouslyinjected into C57BL/6-WT mice. Following tumor growth to 5-10 mm3 involume, mice were treated with dacarbazine (50 mg/kg, i.p., daily) or acontrol vehicle and randomly assigned to regular chow or a chowsupplemented with GW3965 (100 mg/kg). Final day tumor volumemeasurements are shown in (31D). n=8-16 (31B), 7-8 (31C). (31E-31F)Tumor growth by DTIC-sensitive MeWo parental cells and in vivo-derivedDTIC-resistant MeWo human melanoma cells in response to DTIC or GW3965treatments. 5×105 cells were subcutaneously injected into NOD Scid gammamice. After formation of tumors measuring 5-10 mm3 in volume, mice wereblindedly assigned to a control treatment, a DTIC treatment (50 mg/kg,i.p., administered daily in 5-day cycles with 2-day off-treatmentintervals), or a GW3965-supplemented diet treatment (100 mg/kg). Finalday tumor measurements are show in (31F). n=6-8. (31G) Tumor growth by2×106 SK-Mel-239 vemurafenib-resistant clone cells subcutaneouslyinjected into NOD Scid gamma mice that were assigned to a control dietor a diet supplemented with GW3965 (100 mg/kg) subsequent to growth oftumors to 5-10 mm3 in volume. n=7-8. (31H) Overall mouse survivalpost-grafting of 2×106 SK-Mel-239 vemurafenib-resistant cells. Upon thegrowth of tumors to 5-10 mm3 in volume, mice were continuously fed acontrol diet or a diet supplemented with GW3965 (100 mg/kg). n=7. (31I)Experimentally derived model depicting the engagement of systemic andmelanoma-autonomous ApoE by LXRβ activation therapy in mediating thesuppression of melanoma progression phenotypes. Extracellular ApoEsuppresses melanoma metastasis by coordinately inhibiting melanoma cellinvasion and non-cell-autonomous endothelial recruitment throughtargeting melanoma-cell LRP1 and endothelial-cell LRP8 receptors,respectively. All data are represented as mean±SEM. Scale bar, 5 mm.

FIG. 32. Dacarbazine-Induced Suppression of Tumor Growth by HumanMelanoma Cells. Tumor growth by 5×105 DTIC-sensitive MeWo parental cellssubcutaneously injected into Nod SCID gamma mice. When tumors reached5-10 mm3 volume, mice were treated with a control vehicle or DTIC (50mg/kg, i.p., administered daily in 5-day cycles with 2-day off-treatmentintervals), and tumor volume was measured twice a week. n=6.

FIGS. 33A, 33B, 33C, 33D, 33E, 33F, 33G, 33H and I. ApoE-mediatedsuppression of cell invasion across multiple cancer types. (33A-33B)5×104 MUM2B and OCM1 human uveal melanoma cells, (33C-33E) 5×104MDA-231, MDA-468, and BT 549 human triple-negative breast cancer cells,(33F-33G) 5×104 PANC1 and BXPC-3 human pancreatic cancer cells, and(33H-33I) 5×104 786-00 and RCC4 human renal cancer cells were tested fortheir ability to invade through matrigel-coated trans-well inserts invitro. BSA or recombinant ApoE were added to the cell media at 100 m/mLat the start of the assay. n=4. All data are represented as mean±SEM;*p<0.05, **p<0.01, ***p<0.001.

FIGS. 34A, 34B, 34C and 34D. Effects of LXR agonists LXR-623,WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, and SB742881 on ApoEexpression in human melanoma cells. (34A-34D) MeWo human melanoma cellswere treated with DMSO or the LXR agonists LXR-623 (34A), WO-2007-002563(34B), WO-2010-0138598 (34C), or SB742881 (34D) at 500 nM, 1 μM, or 2 μMfor 48 hours. The expression levels of ApoE were subsequently quantifiedby qRT-PCR. n=3. All data are represented as mean±SEM. *p<0.05,**p<0.01.

FIGS. 35A, 35B and 35C. Treatment with the LXR agonist GW3965 inhibitsIn Vitro tumor cell invasion of renal cancer, pancreatic cancer, andlung cancer. (35A, 35B, 35C) Trans-well matrigel invasion by 5×104 RCChuman renal cancer cells (35A), 5×104 PANC1 human pancreatic cancercells (35B), and 5×104 H460 human lung cancer cells (35C) that weretreated with DMSO or GW3965 at 1 μM for 72 hours prior to the assay.n=4. All data are represented as mean±SEM. *p<0.05, **p<0.01.

FIG. 36. Treatment with the LXR agonist GW3965 inhibits breast cancertumor growth In Vivo. Primary tumor growth by 2×106 MDA-468 human breastcancer cells injected into the mammary fat pads of NOD Scid gamma mice.Two days prior to cancer cell injection, the mice were assigned to acontrol diet treatment or a diet supplemented with GW3965 (75 mg/kg) andmaintained on the corresponding diet throughout the experiment. n=8. Alldata are represented as mean±SEM. ***p<0.001.

FIGS. 37A and 37B. Effects of LXR agonists LXR-623, WO-2007-002563 Ex.19, WO-2010-0138598 Ex. 9, and SB742881 on in vitro melanoma progressionphenotypes. (37A) Cell invasion by 1×105 MeWo human melanoma cellspre-treated with DMSO, LXR-623, WO-2007-002563 Ex. 19, WO-2010-0138598Ex. 9, or SB742881 at 1 μM each for 72 hours. The number of cellsinvading into the basal side of matrigel-coated trans-well inserts wasquantified. n=5. (37B) Endothelial recruitment by 5×104 MeWo cellspre-treated with DMSO, LXR-623, WO-2007-002563 Ex. 19, WO-2010-0138598Ex.9, or SB742881 at 1 μM each for 72 hours. Cancer cells were seeded atthe bottom of a 24-well plate. Endothelial cells were seeded in atrans-well insert fitted into each well and allowed to migrate towardsthe cancer cells. The number of endothelial cells migrating to the basalside of each trans-well insert was quantified. n=4-5. All data arerepresented as mean±SEM. *p<0.05, **p<0.01.

FIGS. 38A, 38B, 38C and 38D. Effects of LXR agonists LXR-623,WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, and SB742881 on in vivotumor growth. (38A, 38B, 38C, 38D) Tumor growth by 5×104 B16F10 mousemelanoma cells subcutaneously injected into 7-week-old C57BL/6 mice.After tumors reached 5-10 mm3 in volume, the mice were randomly assignedto a control diet treatment, an LXR-623-supplemented diet treatment at20 mg/kg/day (38A) a WO-2007-002563 Ex. 19-supplemented diet treatmentat 100 mg/kg/day (38B), a WO-2010-0138598 Ex. 19-supplemented diettreatment at 10 mg/kg/day or 100 mg/kg/day (38C), or anSB742881-supplemented diet treatment at 100 mg/kg/day (38D). n=8-10. Alldata are represented as mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods for preventing or reducingaberrant proliferation, differentiation, or survival of cells. Forexample, compounds of the invention may be useful in reducing the riskof, or preventing, tumors from increasing in size or from reaching ametastatic state. The subject compounds may be administered to halt theprogression or advancement of cancer. In addition, the instant inventionincludes use of the subject compounds to reduce the risk of, or prevent,a recurrence of cancer.

Metastatic progression requires that sets of effector proteins involvedin common cellular phenotypes be coherently expressed (Gupta andMassagué, 2006 Cell 127, 679-695; Hanahan and Weinberg, 2011 Cell 144,646-674; Talmadge and Fidler, 2010 Cancer Res. 70, 5649-5669; Hynes,2003 Cell 113, 821-823). Such concerted expression states are apparentin gene expression profiles of primary breast cancers that metastasize(Wang et al., 2005 Lancet 365, 671-679), as well as profiles of humancancer cell clones that display enhanced metastatic activity (Kang etal., 2003 Cancer Cell 3, 537-549; Minn et al., 2005 Nature 436,518-524). In recent years, post-transcriptional regulation has emergedas a pervasive and robust mode of concerted expression-state andphenotype-level control. The most studied class of post-transcriptionalregulators with metastatic regulatory activity are small non-coding RNAs(miRNAs) (Bartel, 2009 Cell 136, 215-233; Fabian et al., 2010 Annu. Rev.Biochem, 79, 351-379; Filipowicz et al., 2008 Nat. Rev. Genet. 9,102-114). Metastasis promoter miRNAs (Ma et al., 2007 Nature 449,682-688; Huang et al., 2008 Nat. Cell Biol. 10, 202-210) and suppressormiRNAs (Tavazoie et al., 2008 Nature 451, 147-152) were originallydiscovered in breast cancer. Subsequent studies revealed many moremiRNAs with regulatory roles in the tumorigenesis and metastasis ofother cancer types (Hatziapostolou et al., 2011 Cell 147, 1233-1247;Hurst et al., 2009 Cancer Res. 69, 7495-7498; Olson et al., 2009 GenesDev. 23, 2152-2165; Zhang et al., 2010 Oncogene 29, 937-948) In manycases, the expression levels of these miRNAs in human cancer sampleshave supported their experimental roles in metastasis. Thus, deregulatedmiRNA expression (Garzon et al., 2010 Nat. Rev. Drug Discov. 9, 775-789;Lujambio and Lowe, 2012 Nature 482, 347-355) and, more recently,deregulated expression of long non-coding RNAs (Calin et al., 2007 Nat.Rev. Cancer 6, 857-866; Gupta et al., 2010 Nature 464, 1071-1076;Guttman et al., 2009 Nature 458, 223-227; Huarte et al., 2010. Cell 142,409-419; Loewer et al., 2010 Nat. Genet. 42, 1113-1117) as well asnon-coding pseudogenes competing for endogenous miRNA binding (Polisenoet al., 2010 Nature 465, 1033-1038) appear to be pervasive features ofhuman cancer. Clues regarding the robust control exerted by specificmiRNAs on metastatic progression came from early work showing thatconcerted targeting of multiple metastasis genes by a single metastasissuppressor miRNA was responsible for the dramatic metastasis suppressioneffects (Tavazoie et al., 2008 Nature 451, 147-152). Such divergent genetargeting by miRNAs has appeared to be a defining feature of theseregulators.

At a conceptual level, the need for divergent regulation of geneexpression in cancer is readily understood. A miRNA could exert robustmetastatic suppression by virtue of its ability to target multiple genesrequired for metastasis. The miRNA's silencing through genetic orepigenetic mechanisms would readily promote cancer progression byde-repressing multiple promoters of metastasis (Png et al., 2011 Nature481, 190-194). A role for convergent regulation of a single gene bymultiple metastasis regulatory miRNAs is more nuanced. This scenariowould emerge if there existed a key gene that acted as a robustsuppressor of metastatic progression. Convergent and cooperativetargeting of this gene by multiple miRNAs could achieve maximalsilencing of such a key metastasis suppressor gene. This scenario, asopposed to genetic deletion, may be seen in cases where complete loss ofa target gene could not be tolerated by the cell, and the gene would berequired at low levels to mediate metabolic actions, for example. Giventhis possibility, a search for cooperative metastasis promoter miRNAsmay uncover novel genes that are pivotal for metastasis suppression andmay provide therapeutic insights into more effective treatments formetastasis prevention.

As disclosed herein, via a systematic, in vivo selection-based approach,a set of miRNAs were identified to be deregulated in multipleindependent metastatic lines derived from multiple patients withmelanoma—a highly prevalent cancer with increasing incidence (Garbe andLeiter, 2009 Clin. Dermatol. 27, 3-9). As disclosed herein, miR-1908,miR-199a-3p, and miR-199a-5p act as robust endogenous promoters ofmelanoma metastasis through convergent targeting of the metabolic geneApoE and the heat-shock protein DNAJA4. Through loss-of-function,gain-of-function, and epistatic analyses, a cooperative miRNA networkthat maximally silences ApoE signaling is delineated. Cancercell-secreted ApoE inhibits metastatic invasion and endothelialrecruitment, which is mediated through its actions on distinct receptorson melanoma and endothelial cells. These miRNAs display significantprognostic capacity in identifying patients that develop melanomametastatic relapse, while therapeutic delivery of LNAs targeting thesemiRNAs significantly inhibits melanoma metastasis. The current lack ofeffective therapies for the prevention of melanoma metastasis aftersurgical resection (Garbe et al., 2011 Oncologist 16, 5-24) requires animproved molecular and mechanistic understanding of melanoma metastaticprogression. To this end, the findings disclosed herein reveal a numberof key novel non-coding and coding genes involved in melanomaprogression and offer a novel avenue for both identifying patients athigh-risk for melanoma metastasis and treating them.

Listed below are the nucleic acid and amino acid sequences of themembers of the above-mentioned network and a number of other sequences.

APOE- RNA sequence (SEQ ID NO: 1)gggatccttgagtcctactcagccccagcggaggtgaaggacgtccttccccaggagccgactggccaatcacaggcaggaagatgaaggttctgtgggctgcgttgctggtcacattcctggcaggatgccaggccaaggtggagcaagcggtggagacagagccggagcccgagctgcgccagcagaccgagtggcagagcggccagcgctgggaactggcactgggtcgcttttgggattacctgcgctgggtgcagacactgtctgagcaggtgcaggaggagctgctcagctcccaggtcacccaggaactgagggcgctgatggacgagaccatgaaggagttgaaggcctacaaatcggaactggaggaacaactgaccccggtggcggaggagacgcgggcacggctgtccaaggagctgcaggcggcgcaggcccggctgggcgcggacatggaggacgtgtgcggccgcctggtgcagtaccgcggcgaggtgcaggccatgctcggccagagcaccgaggagctgcgggtgcgcctcgcctcccacctgcgcaagctgcgtaagcggctcctccgcgatgccgatgacctgcagaagcgcctggcagtgtaccaggccggggcccgcgagggcgccgagcgcggcctcagcgccatccgcgagcgcctggggcccctggtggaacagggccgcgtgcgggccgccactgtgggctccctggccggccagccgctacaggagcgggcccaggcctggggcgagcggctgcgcgcgcggatggaggagatgggcagccggacccgcgaccgcctggacgaggtgaaggagcaggtggcggaggtgcgcgccaagctggaggagcaggcccagcagatacgcctgcaggccgaggccttccaggcccgcctcaagagctggttcgagcccctggtggaagacatgcagcgccagtgggccgggctggtggagaaggtgcaggctgccgtgggcaccagcgccgcccctgtgcccagcgacaatcactgaacgccgaagcctgcagccatgcgaccccacgccaccccgtgcctcctgcctccgcgcagcctgcagcgggagaccctgtccccgccccagccgtcctcctggggtggaccctagtttaataaagattcaccaagtttcacgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa APOE- Amino acid sequence (SEQ ID NO: 2)mkvlwaallv tflagcqakv eqavetepep elrqqtewqsgqrwelalgr fwdylrwvqt lseqvqeell ssqvtqelralmdetmkelk aykseleeql tpvaeetrar lskelqaaqarlgadmedvc grlvqyrgev qamlgqstee lrvrlashlrklrkrllrda ddlqkrlavy qagaregaer glsairerlgplveqgrvra atvgslagqp lqeraqawge rlrarmeemgsrtrdrldev keqvaevrak leeqaqqirl qaeafqarlkswfeplvedm qrqwaglvek vqaavgtsaa pvpsdnh(Underlined residues 136-150 represent the LRP- binding domain of Apo E)DNAJA4 isoform 1- RNA sequence (SEQ ID NO: 3)agucccacccuucggcgcagggcuccggccaacacagcccuccaggccgccuacucuccagccagccggcuccacggacccacggaagggcaagggggcggccucggggcggcgggacaguugucggagggcgcccuccaggcccaagccgccuucuccggcccccgccauggcccggggcggcagucagagcuggagcuccggggaaucagacgggcagccaaaggagcagacgcccgagaagcccagacacaagauggugaaggagacccaguacuaugacauccugggcgugaagcccagcgcguccccggaggagaucaagaaggccuaucggaagcuggcgcucaaguaccacccggacaagaacccggaugagggcgagaaguuuaaacucauaucccaggcauaugaagugcuuucagauccaaagaaaagggauguuuaugaccaaggcggagagcaggcaauuaaagaaggaggcucaggcagccccagcuucucuucacccauggacaucuuugacauguucuuugguggugguggacggauggcuagagagagaagaggcaagaauguuguacaccaguuaucuguaacucuugaagaucuauauaauggagucacgaagaaauuggcccuccagaaaaauguaauuugugagaaaugugaagguguuggugggaagaagggaucgguggagaagugcccgcugugcaaggggcgggggaugcagauccacauccagcagaucgggccgggcaugguacagcagauccagaccgugugcaucgagugcaagggccagggugagcgcaucaaccccaaggaccgcugcgagagcugcagcggggccaaggugauccgugagaagaagauuaucgagguacauguugaaaaagguaugaaagaugggcaaaagauacuauuucauggagaaggagaucaggagccugagcuggagccuggugaugucauaauugugcuugaucagaaggaucauagugucuuucagagacgaggccaugacuugaucaugaaaaugaaaauucagcuuucugaagcucuuuguggcuucaagaagacgauaaaaacauuggacaaucgaauucuuguuauuacauccaaagcaggugaggugauaaagcacggggaccugagaugcgugcgcgaugaaggaaugcccaucuacaaagcaccccuggaaaaagggauucugaucauacaguuuuuaguaaucuuuccugaaaaacacuggcuuucucuggaaaagcuuccucagcuggaagcuuuacucccuccucgacagaaagugaggauuacagaugacauggaucagguggagcugaaggaguuuugucccaaugagcagaacuggcgucagcacagggaggccuacgaggaggacgaagacgggccccaggcuggagugcagugccagacggcaugacguggugcggggcagcguggccccaccggacuagcacaugaugaauguaaaguuggcacaaugaaaaugacaucgcuuuaauggccuuguguuugggauguccuguguauguguucagcauucuuaauugcugagugucuuuuuggcuuuucuuuugguuguaacuuaaguuauagcuuaauuuauauuuaaauguuuuaaguauaaaucaccucuagucugcauauggaaucuguucauuucuauuuucaggauauacuuuugagaugucagugauugcaccaauacuuugugcuucuaguggcuuugccauaauucagugucaccaauaaggcacagcccaguuagcagcuuagccccccuagcaaaccccaaggcacaaagugggcauccugacucaucucuaggucugugguuucuccccucuucccuuggcagaguuauugagggcaugaucucagggcugcuaagauaacauuucugaggauucuagaugauccucuuaaagaauaaaagcacauccguggaucggacauggcugcaugugccugcuuaacagggccaacuuaguuccuacuguucugugcccuucaguggauggaacgugagugucugaucaucucucuuggaaguuuucugaaccuuccaagcucuguggugaggacaaaccaguguuugaaucauaugcugauaacuguuugccugugacccucacaccuuguucuucaggguuuuaaugauuuucuguugacaacuuuugcaaugcuuucccaccaaagugcuuacuuguaaagaaaacuaaauccuucuguguccccggcagccucagugcagcaacagaagccaagggagaaugcugcugguuuggcccauggcacagccagcuucucugaccaguaauccggggugacuugagggucugcaaaggcauagaacuccccaguguuuuccaccucauucucccagauugagcucccuuccaaaggaucguuccucucauugcacagccauauuacaaaggguuuccugcucaagugauguuuugguaagaacuucgcugaguuccacuguggauuacaguuuguauggacuacuacuguaaauuauagcuuguuuggagggauauuagucauuauuuuauucaugacagguagacuacaauucgaacuuaggguuaccucagucuuuagccauuacugcuuauuucuuuuccccaagucacaaaaaacuuguaagcugcuggguuaaagcagaggccaccugucagaucuacccuacccuuauuugguuacauggcaccugagaguuucacucagaccagggaucuuccuuaggagggucaaagugcagaucagaccaugcagguaaggugaaccagcugcacggaccagguucccgcaaaacauugccagcuagugaggcauaauuugcucaaaguauagaaacagcccaccugugcccacuuugaccauuggugaggauagauauaaaaucacuucuuccaacgaagccuaggugaaaaucuauuuauaaauggaccacaacucuggggugucguuuuugugcugugacuuccuaauuauugcuaaagaacuacuguuuaguugguaaugguguaaaauuacauucagcuccuucuugucauauaaaaggaauuuggagggugucgcuuaaaauuuuauuccaccuguacauuugucacuuuaaaauuaaaauugagcugguaugagagauaaaaaaaaaaaaaaaa aaaDNAJA4 isoform 1- Amino acid sequence (SEQ ID NO: 4)marggsqsws sgesdgqpke qtpekprhkm vketqyydilgvkpsaspee ikkayrklal kyhpdknpde gekfldisqayevlsdpkkr dvydqggeqa ikeggsgsps fsspmdifdmffggggrmar errgknvvhq lsvtledlyn gvtkklalqknvicekcegv ggkkgsvekc plckgrgmqi hiqqigpgmvqqiqtvciec kgqgerinpk drcescsgak virekkiievhvekgmkdgq kilfhgegdq epelepgdvi ivldqkdhsvfqrrghdlim kmkiqlseal cgfkktikti dnrilvitskagevikhgdl revrdegmpi ykaplekgil iiqflvifpekhwlsleklp qleallpprq kvritddmdq velkefcpne qnwrqhreay eededgpqag vqcqtaDNAJA4 isoform 2- RNA sequence (SEQ ID NO: 5)gugaccgugacgcgcgagcgggcggcgggggcgcgggccaggggcgcgggccagggugccggcaggggcguccggggcgcucugaccggccucgcccgccccccccgcagacacaagauggugaaggagacccaguacuaugacauccugggcgugaagcccagcgcguccccggaggagaucaagaaggccuaucggaagcuggcgcucaaguaccacccggacaagaacccggaugagggcgagaaguuuaaacucauaucccaggcauaugaagugcuuucagauccaaagaaaagggauguuuaugaccaaggcggagagcaggcaauuaaagaaggaggcucaggcagccccagcuucucuucacccauggacaucuuugacauguucuuugguggugguggacggauggcuagagagagaagaggcaagaauguuguacaccaguuaucuguaacucuugaagaucuauauaauggagucacgaagaaauuggcccuccagaaaaauguaauuugugagaaaugugaagguguuggugggaagaagggaucgguggagaagugcccgcugugcaaggggcgggggaugcagauccacauccagcagaucgggccgggcaugguacagcagauccagaccgugugcaucgagugcaagggccagggugagcgcaucaaccccaaggaccgcugcgagagcugcagcggggccaaggugauccgugagaagaagauuaucgagguacauguugaaaaagguaugaaagaugggcaaaagauacuauuucauggagaaggagaucaggagccugagcuggagccuggugaugucauaauugugcuugaucagaaggaucauagugucuuucagagacgaggccaugacuugaucaugaaaaugaaaauucagcuuucugaagcucuuuguggcuucaagaagacgauaaaaacauuggacaaucgaauucuuguuauuacauccaaagcaggugaggugauaaagcacggggaccugagaugcgugcgcgaugaaggaaugcccaucuacaaagcaccccuggaaaaagggauucugaucauacaguuuuuaguaaucuuuccugaaaaacacuggcuuucucuggaaaagcuuccucagcuggaagcuuuacucccuccucgacagaaagugaggauuacagaugacauggaucagguggagcugaaggaguuuugucccaaugagcagaacuggcgucagcacagggaggccuacgaggaggacgaagacgggccccaggcuggagugcagugccagacggcaugacguggugcggggcagcguggccccaccggacuagcacaugaugaauguaaaguuggcacaaugaaaaugacaucgcuuuaauggccuuguguuugggauguccuguguauguguucagcauucuuaauugcugagugucuuuuuggcuuuucuuuugguuguaacuuaaguuauagcuuaauuuauauuuaaauguuuuaaguauaaaucaccucuagucugcauauggaaucuguucauuucuauuuucaggauauacuuuugagaugucagugauugcaccaauacuuugugcuucuaguggcuuugccauaauucagugucaccaauaaggcacagcccaguuagcagcuuagccccccuagcaaaccccaaggcacaaagugggcauccugacucaucucuaggucugugguuucuccccucuucccuuggcagaguuauugagggcaugaucucagggcugcuaagauaacauuucugaggauucuagaugauccucuuaaagaauaaaagcacauccguggaucggacauggcugcaugugccugcuuaacagggccaacuuaguuccuacuguucugugcccuucaguggauggaacgugagugucugaucaucucucuuggaaguuuucugaaccuuccaagcucuguggugaggacaaaccaguguuugaaucauaugcugauaacuguuugccugugacccucacaccuuguucuucaggguuuuaaugauuuucuguugacaacuuuugcaaugcuuucccaccaaagugcuuacuuguaaagaaaacuaaauccuucuguguccccggcagccucagugcagcaacagaagccaagggagaaugcugcugguuuggcccauggcacagccagcuucucugaccaguaauccggggugacuugagggucugcaaaggcauagaacuccccaguguuuuccaccucauucucccagauugagcucccuuccaaaggaucguuccucucauugcacagccauauuacaaaggguuuccugcucaagugauguuuugguaagaacuucgcugaguuccacuguggauuacaguuuguauggacuacuacuguaaauuauagcuuguuuggagggauauuagucauuauuuuauucaugacagguagacuacaauucgaacuuaggguuaccucagucuuuagccauuacugcuuauuucuuuuccccaagucacaaaaaacuuguaagcugcuggguuaaagcagaggccaccugucagaucuacccuacccuuauuugguuacauggcaccugagaguuucacucagaccagggaucuuccuuaggagggucaaagugcagaucagaccaugcagguaaggugaaccagcugcacggaccagguucccgcaaaacauugccagcuagugaggcauaauuugcucaaaguauagaaacagcccaccugugcccacuuugaccauuggugaggauagauauaaaaucacuucuuccaacgaagccuaggugaaaaucuauuuauaaauggaccacaacucuggggugucguuuuugugcugugacuuccuaauuauugcuaaagaacuacuguuuaguugguaaugguguaaaauuacauucagcuccuucuugucauauaaaaggaauuuggagggugucgcuuaaaauuuuauuccaccuguacauuugucacuuuaaaauuaaaauugagcugguaugagagauaaaaa aaaaaaaaaaaaaaDNAJA4 isoform 2- Amino acid sequence (SEQ ID NO: 6)mvketqyydi lgvkpsaspe eikkayrkla lkyhpdknpdegekfklisq ayevlsdpkk rdvydqggeq aikeggsgspsfsspmdifd mffggggrma rerrgknvvh qlsvtledlyngvtkklalq knvicekceg vggkkgsvek cplckgrgmqihiqqigpgm vqqiqtvcie ckgqgerinp kdrcescsgakvirekkiie vhvekgmkdg qkilfhgegd qepelepgdviivldqkdhs vfqrrghdli mkmkiqlsea lcgfkktiktldnrilvits kagevikhgd lrcvrdegmp iykaplekgiliiqflvifp ekhwlslekl pqleallppr qkvritddmdqvelkefcpn eqnwrqhrea yeededgpqa gvqcqta DNAJA4 isoform 3- RNA sequence(SEQ ID NO: 7) acauuucagcaagcuggcuaaagacaugugggaaagccugacccuggauucaggucaaaucucagcacucacaagauuuaaacucauaucccaggcauaugaagugcuuucagauccaaagaaaagggauguuuaugaccaaggcggagagcaggcaauuaaagaaggaggcucaggcagccccagcuucucuucacccauggacaucuuugacauguucuuugguggugguggacggauggcuagagagagaagaggcaagaauguuguacaccaguuaucuguaacucuugaagaucuauauaauggagucacgaagaaauuggcccuccagaaaaauguaauuugugagaaaugugaagguguuggugggaagaagggaucgguggagaagugcccgcugugcaaggggcgggggaugcagauccacauccagcagaucgggccgggcaugguacagcagauccagaccgugugcaucgagugcaagggccagggugagcgcaucaaccccaaggaccgcugcgagagcugcagcggggccaaggugauccgugagaagaagauuaucgagguacauguugaaaaagguaugaaagaugggcaaaagauacuauuucauggagaaggagaucaggagccugagcuggagccuggugaugucauaauugugcuugaucagaaggaucauagugucuuucagagacgaggccaugacuugaucaugaaaaugaaaauucagcuuucugaagcucuuuguggcuucaagaagacgauaaaaacauuggacaaucgaauucuuguuauuacauccaaagcaggugaggugauaaagcacggggaccugagaugcgugcgcgaugaaggaaugcccaucuacaaagcaccccuggaaaaagggauucugaucauacaguuuuuaguaaucuuuccugaaaaacacuggcuuucucuggaaaagcuuccucagcuggaagcuuuacucccuccucgacagaaagugaggauuacagaugacauggaucagguggagcugaaggaguuuugucccaaugagcagaacuggcgucagcacagggaggccuacgaggaggacgaagacgggccccaggcuggagugcagugccagacggcaugacguggugcggggcagcguggccccaccggacuagcacaugaugaauguaaaguuggcacaaugaaaaugacaucgcuuuaauggccuuguguuugggauguccuguguauguguucagcauucuuaauugcugagugucuuuuuggcuuuucuuuugguuguaacuuaaguuauagcuuaauuuauauuuaaauguuuuaaguauaaaucaccucuagucugcauauggaaucuguucauuucuauuuucaggauauacuuuugagaugucagugauugcaccaauacuuugugcuucuaguggcuuugccauaauucagugucaccaauaaggcacagcccaguuagcagcuuagccccccuagcaaaccccaaggcacaaagugggcauccugacucaucucuaggucugugguuucuccccucuucccuuggcagaguuauugagggcaugaucucagggcugcuaagauaacauuucugaggauucuagaugauccucuuaaagaauaaaagcacauccguggaucggacauggcugcaugugccugcuuaacagggccaacuuaguuccuacuguucugugcccuucaguggauggaacgugagugucugaucaucucucuuggaaguuuucugaaccuuccaagcucuguggugaggacaaaccaguguuugaaucauaugcugauaacuguuugccugugacccucacaccuuguucuucaggguuuuaaugauuuucuguugacaacuuuugcaaugcuuucccaccaaagugcuuacuuguaaagaaaacuaaauccuucuguguccccggcagccucagugcagcaacagaagccaagggagaaugcugcugguuuggcccauggcacagccagcuucucugaccaguaauccggggugacuugagggucugcaaaggcauagaacuccccaguguuuuccaccucauucucccagauugagcucccuuccaaaggaucguuccucucauugcacagccauauuacaaaggguuuccugcucaagugauguuuugguaagaacuucgcugaguuccacuguggauuacaguuuguauggacuacuacuguaaauuauagcuuguuuggagggauauuagucauuauuuuauucaugacagguagacuacaauucgaacuuaggguuaccucagucuuuagccauuacugcuuauuucuuuuccccaagucacaaaaaacuuguaagcugcuggguuaaagcagaggccaccugucagaucuacccuacccuuauuugguuacauggcaccugagaguuucacucagaccagggaucuuccuuaggagggucaaagugcagaucagaccaugcagguaaggugaaccagcugcacggaccagguucccgcaaaacauugccagcuagugaggcauaauuugcucaaaguauagaaacagcccaccugugcccacuuugaccauuggugaggauagauauaaaaucacuucuuccaacgaagccuaggugaaaaucuauuuauaaauggaccacaacucuggggugucguuuuugugcugugacuuccuaauuauugcuaaagaacuacuguuuaguugguaaugguguaaaauuacauucagcuccuucuugucauauaaaaggaauuuggagggugucgcuuaaaauuuuauuccaccuguacauuugucacuuuaaaauuaaaauugagcugguaugagagauaaaaaaaaaaaaaaaaaaaDNAJA4 isoform 3- Amino acid sequence (SEQ ID NO: 8)mwesltldsg qisaltrfkl isqayevlsd pkkrdvydqggeqaikeggs gspsfsspmd ifdmffgggg rmarerrgknvvhqlsvtle dlyngvtkkl alqknvicek cegvggkkgsvekcplckgr gmqihiqqig pgmvqqiqtv cieckgqgerinpkdrcesc sgakvirekk iievhvekgm kdgqkilfhgegdqepelep gdviivldqk dhsvfqrrgh dlimkmkiqlsealcgfkkt iktldnrilv itskagevik hgdlrcvrdegmpiykaple kgiliiqflv ifpekhwlsl eklpqleallpprqkvritd dmdqvelkef cpneqnwrqh reayeededg pqagvqcqtaLRP1- RNA sequence (SEQ ID NO: 9)cagcggugcgagcuccaggcccaugcacugaggaggcggaaacaaggggagcccccagagcuccaucaagcccccuccaaaggcuccccuacccgguccacgccccccacccccccuccccgccuccucccaauugugcauuuuugcagccggaggcggcuccgagauggggcugugagcuucgcccggggagggggaaagagcagcgaggagugaagcggggggguggggugaaggguuuggauuucggggcagggggcgcacccccgucagcaggcccuccccaaggggcucggaacucuaccucuucacccacgccccuggugcgcuuugccgaaggaaagaauaagaacagagaaggaggagggggaaaggaggaaaagggggaccccccaacuggggggggugaaggagagaaguagcaggaccagaggggaaggggcugcugcuugcaucagcccacaccaugcugaccccgccguugcuccugcugcugccccugcucucagcucuggucgcggcggcuaucgacgccccuaagacuugcagccccaagcaguuugccugcagagaucaaauaaccuguaucucaaagggcuggcggugcgacggugagagggacugcccagacggaucugacgaggccccugagauuuguccacagaguaaggcccagcgaugccagccaaacgagcauaacugccuggguacugagcuguguguucccaugucccgccucugcaaugggguccaggacugcauggacggcucagaugaggggccccacugccgagagcuccaaggcaacugcucucgccugggcugccagcaccauuguguccccacacucgaugggcccaccugcuacugcaacagcagcuuucagcuucaggcagauggcaagaccugcaaagauuuugaugagugcucaguguacggcaccugcagccagcuaugcaccaacacagacggcuccuucauauguggcuguguugaaggauaccuccugcagccggauaaccgcuccugcaaggccaagaacgagccaguagaccggcccccugugcuguugauagccaacucccagaacaucuuggccacguaccugaguggggcccaggugucuaccaucacaccuacgagcacgcggcagaccacagccauggacuucagcuaugccaacgagaccguaugcugggugcauguuggggacagugcugcucagacgcagcucaagugugcccgcaugccuggccuaaagggcuucguggaugagcacaccaucaacaucucccucagucugcaccacguggaacagauggccaucgacuggcugacaggcaacuucuacuuuguggaugacaucgaugauaggaucuuugucugcaacagaaauggggacacaugugucacauugcuagaccuggaacucuacaaccccaagggcauugcccuggacccugccauggggaagguguuuuucacugacuaugggcagaucccaaagguggaacgcugugacauggaugggcagaaccgcaccaagcucgucgacagcaagauuguguuuccucauggcaucacgcuggaccuggucagccgccuugucuacugggcagaugccuaucuggacuauauugaagugguggacuaugagggcaagggccgccagaccaucauccagggcauccugauugagcaccuguacggccugacuguguuugagaauuaucucuaugccaccaacucggacaaugccaaugcccagcagaagacgagugugauccgugugaaccgcuuuaacagcaccgaguaccagguugucacccggguggacaaggguggugcccuccacaucuaccaccagaggcgucagccccgagugaggagccaugccugugaaaacgaccaguaugggaagccggguggcugcucugacaucugccugcuggccaacagccacaaggcgcggaccugccgcugccguuccggcuucagccugggcagugacgggaagucaugcaagaagccggagcaugagcuguuccucguguauggcaagggccggccaggcaucauccggggcauggauaugggggccaaggucccggaugagcacaugauccccauugaaaaccucaugaacccccgagcccuggacuuccacgcugagaccggcuucaucuacuuugccgacaccaccagcuaccucauuggccgccagaagauugauggcacugagcgggagaccauccugaaggacggcauccacaauguggaggguguggccguggacuggaugggagacaaucuguacuggacggacgaugggcccaaaaagacaaucagcguggccaggcuggagaaagcugcucagacccgcaagacuuuaaucgagggcaaaaugacacaccccagggcuauugugguggauccacucaauggguggauguacuggacagacugggaggaggaccccaaggacagucggcgugggcggcuggagagggcguggauggauggcucacaccgagacaucuuugucaccuccaagacagugcuuuggcccaaugggcuaagccuggacaucccggcugggcgccucuacuggguggaugccuucuacgaccgcaucgagacgauacugcucaauggcacagaccggaagauuguguaugaagguccugagcugaaccacgccuuuggccugugucaccauggcaacuaccucuucuggacugaguaucggaguggcagugucuaccgcuuggaacgggguguaggaggcgcaccccccacugugacccuucugcgcagugagcggccccccaucuuugagauccgaauguaugaugcccagcagcagcaaguuggcaccaacaaaugccgggugaacaauggcggcugcagcagccugugcuuggccaccccugggagccgccagugcgccugugcugaggaccagguguuggacgcagacggcgucacuugcuuggcgaacccauccuacgugccuccaccccagugccagccaggcgaguuugccugugccaacagccgcugcauccaggagcgcuggaagugugacggagacaacgauugccuggacaacagugaugaggccccagcccucugccaucagcacaccugccccucggaccgauucaagugcgagaacaaccggugcauccccaaccgcuggcucugcgacggggacaaugacugugggaacagugaagaugaguccaaugccacuuguucagcccgcaccugcccccccaaccaguucuccugugccaguggccgcugcauccccaucuccuggacgugugaucuggaugacgacuguggggaccgcucugaugagucugcuucgugugccuaucccaccugcuucccccugacucaguuuaccugcaacaauggcagauguaucaacaucaacuggagaugcgacaaugacaaugacuguggggacaacagugacgaagccggcugcagccacuccuguucuagcacccaguucaagugcaacagcgggcguugcauccccgagcacuggaccugcgauggggacaaugacugcggagacuacagugaugagacacacgccaacugcaccaaccaggccacgaggcccccugguggcugccacacugaugaguuccagugccggcuggauggacuaugcaucccccugcgguggcgcugcgauggggacacugacugcauggacuccagcgaugagaagagcugugagggagugacccacgucugcgaucccagugucaaguuuggcugcaaggacucagcucggugcaucagcaaagcgugggugugugauggcgacaaugacugugaggauaacucggacgaggagaacugcgagucccuggccugcaggccacccucgcacccuugugccaacaacaccucagucugccugcccccugacaagcugugugauggcaacgacgacuguggcgacggcucagaugagggcgagcucugcgaccagugcucucugaauaacgguggcugcagccacaacugcucaguggcaccuggcgaaggcauuguguguuccugcccucugggcauggagcuggggcccgacaaccacaccugccagauccagagcuacugugccaagcaucucaaaugcagccaaaagugcgaccagaacaaguucagcgugaagugcuccugcuacgagggcuggguccuggaaccugacggcgagagcugccgcagccuggaccccuucaagccguucaucauuuucuccaaccgccaugaaauccggcgcaucgaucuucacaaaggagacuacagcguccuggugcccggccugcgcaacaccaucgcccuggacuuccaccucagccagagcgcccucuacuggaccgacgugguggaggacaagaucuaccgcgggaagcugcuggacaacggagcccugacuaguuucgagguggugauucaguauggccuggccacacccgagggccuggcuguagacuggauugcaggcaacaucuacuggguggagaguaaccuggaucagaucgagguggccaagcuggaugggacccuccggaccacccugcuggccggugacauugagcacccaagggcaaucgcacuggauccccgggaugggauccuguuuuggacagacugggaugccagccugccccgcauugaggcagccuccaugaguggggcugggcgccgcaccgugcaccgggagaccggcucugggggcuggcccaacgggcucaccguggacuaccuggagaagcgcauccuuuggauugacgccaggucagaugccauuuacucagcccguuacgacggcucuggccacauggaggugcuucggggacacgaguuccugucgcacccguuugcagugacgcuguacgggggggaggucuacuggacugacuggcgaacaaacacacuggcuaaggccaacaagug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gcauggauggguccagccgcagcgucaucguggacaccaagaucacauggcccaauggccugacgcuggacuaugucacugagcgcaucuacugggccgacgcccgcgaggacuacauugaauuugccagccuggauggcuccaaucgccacguugugcugagccaggacaucccgcacaucuuugcacugacccuguuugaggacuacgucuacuggaccgacugggaaacaaaguccauuaaccgagcccacaagaccacgggcaccaacaaaacgcuccucaucagcacgcugcaccggcccauggaccugcaugucuuccaugcccugcgccagccagacgugcccaaucaccccugcaaggucaacaaugguggcugcagcaaccugugccugcugucccccgggggagggcacaaaugugccugccccaccaacuucuaccugggcagcgaugggcgcaccuguguguccaacugcacggcuagccaguuuguaugcaagaacgacaagugcauccccuucugguggaagugugacaccgaggacgacugcggggaccacucagacgagcccccggacugcccugaguucaagugccggcccggacaguuccagugcuccacagguaucugcacaaacccugccuucaucugcgauggcgacaaugacugccaggacaacagugacgaggccaacugugacauccacgucugcuugcccagucaguucaaaugcaccaacaccaaccgcuguauucccggcaucuuccgcugcaaugggcaggacaacugcggagauggggaggaugagagggacugccccgaggugaccugcgcccccaaccaguuccagugcuccauuaccaaacggugcaucccccgggucugggucugcgaccgggacaaugacuguguggauggcagugaugagcccgccaacugcacccagaugaccugugguguggacgaguuccgcugcaaggauucgggccgcugcaucccagcgcguuggaagugugacggagaggaugacuguggggauggcucggaugagcccaaggaagagugugaugaacgcaccugugagccauaccaguuccgcugcaagaacaaccgcugcgugcccggccgcuggcagugcgacuacgacaacgauugcggugacaacuccgaugaagagagcugcaccccucggcccugcuccgagagugaguucuccugugccaacggccgcugcaucgcggggcgcuggaaaugcgauggagaccacgacugcgcggacggcucggacgagaaagacugcaccccccgcugugacauggaccaguuccagugcaagagcggccacugcaucccccugcgcuggcgcugugacgcagacgccgacugcauggacggcagcgacgaggaggccugcggcacuggcgugcggaccugcccccuggacgaguuccagugcaacaacaccuugugcaagccgcuggccuggaagugcgauggcgaggaugacuguggggacaacucagaugagaaccccgaggagugugcccgguucgugugcccucccaaccggcccuuccguugcaagaaugaccgcgucugucuguggaucgggcgccaaugcgauggcacggacaacuguggggaugggacugaugaagaggacugugagccccccacagcccacaccacccacugcaaagacaagaaggaguuucugugccggaaccagcgcugccucuccuccucccugcgcugcaacauguucgaugacugcggggacggcucugacgaggaggacugcagcaucgaccccaagcugaccagcugcgccaccaaugccagcaucuguggggacgaggcacgcugcgugcgcaccgagaaagcggccuacugugccugccgcucgggcuuccacaccgugcccggccagcccggaugccaagacaucaacgagugccugcgcuucggcaccugcucccagcucugcaacaacaccaagggcggccaccucugcagcugcgcucggaacuucaugaagacgcacaacaccugcaaggccgaaggcucugaguaccagguccuguacaucgcugaugacaaugagauccgcagccuguuccccggccacccccauucggcuuacgagcaggcauuccagggugacgagaguguccgcauugaugcuauggauguccaugucaaggcuggccgugucuauuggaccaacuggcacacgggcaccaucuccuaccgcagccugccaccugcugcgccuccuaccacuuccaaccgccaccggcgacagauugaccggggugucacccaccucaacauuucagggcugaagaugcccagaggcaucgccaucgacuggguggccggaaacguguacuggaccgacucgggccgagaugugauugagguggcgcagaugaagggcgagaaccgcaagacgcucaucucgggcaugauugacgagccccacgccauugugguggacccacugagggggaccauguacuggucagacuggggcaaccaccccaagauugagacggcagcgauggaugggacgcuucgggagacacuggugcaggacaacauucaguggcccacaggccuggccguggauuaucacaaugagcggcuguacugggcagacgccaagcuuucagucaucggcagcauccggcucaauggcacggaccccauuguggcugcugacagcaaacgaggccuaagucaccccuucagcaucgacgucuuugaggauuacaucuauggugucaccuacaucaauaaucgugucuucaagauccauaaguuuggccacagccccuuggucaaccugacagggggccugagccacgccucugacgugguccuuuaccaucagcacaagcagcccgaagugaccaacccaugugaccgcaagaaaugcgaguggcucugccugcugagccccagugggccugucugcaccugucccaaugggaagcggcuggacaacggcacaugcgugccugugcccucuccaacgccccccccagaugcuccccggccuggaaccuguaaccugcagugcuucaacgguggcagcuguuuccucaaugcacggaggcagcccaagugccgcugccaaccccgcuacacgggugacaagugugaacuggaccagugcugggagcacugucgcaaugggggcaccugugcugccucccccucuggcaugcccacgugccggugccccacgggcuucacgggccccaaaugcacccagcaggugugugcgggcuacugugccaacaacagcaccugcacugucaaccagggcaaccagccccagugccgaugccuacccggcuuccugggcgaccgcugccaguaccggcagugcucuggcuacugugagaacuuuggcacaugccagauggcugcugauggcucccgacaaugccgcugcacugccuacuuugagggaucgaggugugaggugaacaagugcagccgcugucucgaaggggccuguguggucaacaagcagaguggggaugucaccugcaacugcacggauggccggguggcccccagcugucugaccugcgucggccacugcagcaauggcggcuccuguaccaugaacagcaaaaugaugccugagugccagugcccaccccacaugacagggccccggugugaggagcacgucuucagccagcagcagccaggacauauagccuccauccuaaucccucugcuguugcugcugcugcugguucugguggccggagugguauucugguauaagcggcgaguccaaggggcuaagggcuuccagcaccaacggaugaccaacggggccaugaacguggagauuggaaaccccaccuacaagauguacgaaggcggagagccugaugaugugggaggccuacuggacgcugacuuugcccuggacccugacaagcccaccaacuucaccaaccccguguaugccacacucuacauggggggccauggcagucgccacucccuggccagcacggacgagaagcgagaacuccugggccggggcccugaggacgagauaggggaccccuuggcauagggcccugccccgucggacugcccccagaaagccuccugcccccugccggugaaguccuucagugagccccuccccagccagcccuucccuggccccgccggauguauaaauguaaaaaugaaggaauuacauuuuauaugugagcgagcaagccggcaagcgagcacaguauuauuucuccauccccucccugccugcuccuuggcacccccaugcugccuucagggagacaggcagggagggcuuggggcugcaccuccuacccucccaccagaacgcaccccacugggagagcugguggugcagccuuccccucccuguauaagacacuuugccaaggcucuccccucucgccccaucccugcuugcccgcucccacagcuuccugagggcuaauucugggaagggagaguucuuugcugccccugucuggaagacguggcucugggugagguaggcgggaaaggauggaguguuuuaguucuugggggaggccaccccaaaccccagccccaacuccaggggcaccuaugagauggccaugcucaaccccccucccagacaggcccucccugucuccagggcccccaccgagguucccagggcuggagacuuccucugguaaacauuccuccagccuccccuccccuggggacgccaaggaggugggccacacccaggaagggaaagcgggcagccccguuuuggggacgugaacguuuuaauaauuuuugcugaauuccuuuacaacuaaauaacacagauauuguuauaaauaaaauuguaaaaaaaaaaaaaa aaaaaLRP1- Amino acid sequence (SEQ ID NO: 10)mltpplllll pllsalvaaa idapktcspk qfacrdqitciskgwrcdge rdcpdgsdea peicpqskaq rcqpnehnclgtelcvpmsr lcngvqdcmd gsdegphcre lqgncsrlgcqhhcvptldg ptcycnssfq lqadgktckd fdecsvygtcsqlctntdgs ficgcvegyl lqpdnrscka knepvdrppvlliansqnil atylsgaqvs titptstrqt tamdfsyanetvcwvhvgds aaqtqlkcar mpglkgfvde htinislslhhveqmaidwl tgnfyfvddi ddrifvenrn gdtcvtlldlelynpkgial dpamgkvfft dygqipkver cdmdgqnrtklvdskivfph gitldlvsrl vywadayldy ievvdyegkgrqtiiqgili ehlygltvfe nylyatnsdn anaqqktsvirvnrfnstey qvvtrvdkgg alhiyhqrrq prvrshacendqygkpggcs dicllanshk artcrcrsgf slgsdgksckkpehelflvy gkgrpgiirg mdmgakvpde hmipienlmnpraldfhaet gfiyfadtts yligrqkidg teretilkdgihnvegvavd wmgdnlywtd dgpkktisva rlekaaqtrktliegkmthp raivvdping wmywtdweed pkdsrrgrlerawmdgshrd ifytsktylw pnglsldipa grlywydafydrietillng tdrkivyegp elnhafglch hgnylfwteyrsgsvyrler gvggapptvt lirserppif eirmydaqqqqvgtnkcrvn nggcsslcla tpgsrqcaca edqvldadgvtclanpsyvp ppqcqpgefa cansrciqer wkcdgdndcldnsdeapalc hqhtcpsdrf kcennrcipn rwlcdgdndcgnsedesnat csartcppnq fscasgrcip iswtcdldddcgdrsdesas cayptcfplt qftcnngrci ninwrcdndndcgdnsdeag cshscsstqf kcnsgrcipe hwtcdgdndcgdysdethan ctnqatrppg gchtdefqcr ldglciplrwrcdgdtdcmd ssdekscegv thvcdpsvkf gckdsarciskawvcdgdnd cednsdeenc eslacrppsh pcanntsyclppdklcdgnd dcgdgsdege lcdqcslnng gcshncsvapgegivcscpl gmelgpdnht cqiqsycakh lkcsqkcdqnkfsvkcscye gwvlepdges crsldpfkpf iifsnrheirridlhkgdys vlvpglrnti aldfhlsqsa lywtdvvedkiyrgklldng altsfevviq yglatpegla vdwiagniywvesnldqiev akldgtirtt llagdiehpr aialdprdgilfwtdwdasl prieaasmsg agrrtvhret gsggwpngltvdylekrilw idarsdaiys arydgsghme vlrghefishpfavtlygge vywtdwrtnt lakankwtgh nytyvqrtntqpfdlqvyhp srqpmapnpc eanggqgpcs hiclinynrtvscacphlmk lhkdnttcye fkkfflyarq meirgvdldapyynyiisft vpdidnytvl dydareqrvy wsdvrtqaikrafingtgve tvvsadlpna hglavdwvsr nlfwtsydtnkkqinvarld gsfknavvqg leqphglvvh plrgklywtdgdnismanmd gsnrtllfsg qkgpvglaid fpesklywissgnhtinrcn ldgsglevid amrsqlgkat alaimgdklwwadqvsekmg tcskadgsgs vvirnsttly mhmkvydesiqldhkgtnpc synngdcsql clptsettrs cmctagyslrsgqqacegvg sfllysvheg irgipldpnd ksdalvpvsgtslavgidfh aendtiywvd mglstisrak rdqtwredvytngigrvegi avdwiagniy wtdqgfdvie varingsfryvvisqgldkp raitvhpekg ylfwtewgqy priersrldgtervylvnys iswpngisvd yqdgklywcd artdkieridletgenrevy lssnnmdmfs vsvfedfiyw sdrthangsikrgskdnatd svplrtgigy qlkdikvfnr drqkgtnvcavanggcqqlc lyrgrgqrac acahgmlaed gascreyagyllysertilk sihlsdernl napvqpfedp ehmknvialafdyragtspg tpnriffsdi hfgniqqind dgsrritivenvgsveglay hrgwdtlywt syttstitrh tvdqtrpgaferetvitmsg ddhprafvld ecqnlmfwtn wneqhpsimraalsganylt liekdirtpn glaidhraek lyfsdatldkierceydgsh ryvilksepv hpfglavyge hifwtdwvrravqrankhvg snmkllrvdi pqqpmgiiav andtnscelsperinnggcq dlcllthqgh vncscrggri lqddltcravnsscraqdef ecangecinf sltcdgvphc kdksdekpsycnsrrckktf rqcsngrcvs nmlwcngadd cgdgsdeipcnktacgvgef rcrdgtcign ssrcnqfvdc edasdemncsatdcssyfrl gykgylfqpc ertslcyaps wvcdgandcgdysderdcpg vkrprcpiny facpsgrcip mswtcdkeddcehgedethc nkfcseaqfe cqnhrciskq wlcdgsddcgdgsdeaahce gktcgpssfs cpgthycype rwlcdgdkdcadgadesiaa gclynstcdd refmcqnrqc ipkhfvcdhdrdcadgsdes peceyptcgp sefrcangrc lssrqwecdgendchdqsde apknphctsq ehkcnassqf lcssgrcvaeallcngqddc gdssdergch ineclsrkls gcsqdcedlkigfkercrpg frlkddgrtc advdecsttf pcsqrcinthgsykcicveg yaprggdphs ckavtdeepf lifanryylrklnldgsnyt llkqglnnav aldfdyreqm iywtdvttqgsmirrmhlng snvqvlhrtg lsnpdglavd wvggnlywcdkgrdtievsk ingayrtylv ssglrepral vvdvqngylywtdwgdhsli grigmdgssr svivdtkitw pngltldyvteriywadare dyiefasldg snrhyvlsqd iphifaltlfedyvywtdwe tksinrahkt tgtnktllis tlhrpmdlhvfhalrqpdvp nhpckvnngg csnlcllspg gghkcacptnfylgsdgrtc vsnctasqfv ckndkcipfw wkcdteddcgdhsdeppdcp efkcrpgqfq cstgictnpa ficdgdndcqdnsdeancdi hvclpsqfkc tntnrcipgi frcngqdncgdgederdcpe vtcapnqfqc sitkrcipry wvcdrdndcvdgsdepanct qmtcgvdefr ckdsgrcipa rwkcdgeddcgdgsdepkee cdertcepyq frcknnrcvp grwqcdydndcgdnsdeesc tprpcsesef scangrciag rwkcdgdhdcadgsdekdct prcdmdqfqc ksghciplrw rcdadadcmdgsdeeacgtg vrtcpldefq cnntickpla wkcdgeddcgdnsdenpeec arfvcppnrp frckndrycl wigrqcdgtdncgdgtdeed cepptahtth ckdkkeficr nqrclssslrcnmfddcgdg sdeedcsidp kltscatnas icgdearcvrtekaaycacr sgfhtvpgqp gcqdineclr fgtcsqlcnntkgghlcsca rnfmkthntc kaegseyqvl yiaddneirslfpghphsay eqafqgdesv ridamdvhvk agrvywtnwhtgtisyrslp paappttsnr hrrqidrgvt hlnisglkmprgiaidwvag nvywtdsgrd vievaqmkge nrktlisgmidephaivvdp lrgtmywsdw gnhpkietaa mdgtlretlyqdniqwptgl avdyhnerly wadaklsvig siringtdpivaadskrgls hpfsidvfed yiygvtyinn rvfkihkfghsplvnitggl shasdvvlyh qhkqpevtnp cdrkkcewlcllspsgpvct cpngkrldng tcypvpsptp ppdaprpgtcnlqcfnggsc flnarrqpkc rcqprytgdk celdqcwehcrnggtcaasp sgmptcrept gftgpkctqq vcagycannstctvnqgnqp qcrclpgflg drcqyrqcsg ycurlicqnlaadgsrqcrc tayfegsrce vnkcsrcleg acvvnkqsgdvtcnctdgry apscltcvgh csnggsctmn skmmpecqcpphmtgprcee hvfsqqqpgh iasiliplll llllvlvagvvfwykrrvqg akgfqhqrmt ngamnveign ptykmyeggepddvgglida dfaldpdkpt nftnpvyatl ymgghgsrhs lastdekrel lgrgpedeig dplaLRP8 isoform 1- RNA sequence (SEQ ID NO: 11)gcuggcggcggccgcccagggccggggccgcgcgcccagccugagcccgccccgccgccgagcgucaccgaaccugcuugaaaugcagccgaggagccggggcgggcggcagcggcggcggcggcggcggcgggggcagcggcaaccccggcgccgcggcaaggacucggagggcugagacgcggcggcggcggcgcggggagcgcggggcgcggcggccggagccccgggcccgccaugggccuccccgagccgggcccucuccggcuucuggcgcugcugcugcugcugcugcugcugcugcugcugcagcuccagcaucuugcggcggcagcggcugauccgcugcucggcggccaagggccggccaaggauugcgaaaaggaccaauuccagugccggaacgagcgcugcauccccucuguguggagaugcgacgaggacgaugacugcuuagaccacagcgacgaggacgacugccccaagaagaccugugcagacagugacuucaccugugacaacggccacugcauccacgaacgguggaagugugacggcgaggaggaguguccugauggcuccgaugaguccgaggccacuugcaccaagcagguguguccugcagagaagcugagcuguggacccaccagccacaaguguguaccugccucguggcgcugcgacggggagaaggacugcgaggguggagcggaugaggccggcugugcuaccuugugcgccccgcacgaguuccagugcggcaaccgcucgugccuggccgccguguucgugugcgacggcgacgacgacuguggugacggcagcgaugagcgcggcugugcagacccggccugcgggccccgcgaguuccgcugcggcggcgauggcggcggcgccugcaucccggagcgcugggucugcgaccgccaguuugacugcgaggaccgcucggacgaggcagccgagcucugcggccguccgggccccggggccacguccgcgcccgccgccugcgccaccgccucccaguucgccugccgcagcggcgagugcgugcaccugggcuggcgcugcgacggcgaccgcgacugcaaagacaaaucggacgaggccgacugcccacugggcaccugccguggggacgaguuccaguguggggaugggacauguguccuugcaaucaagcacugcaaccaggagcaggacuguccagaugggagugaugaagcuggcugccuacaggggcugaacgagugucugcacaacaauggcggcugcucacacaucugcacugaccucaagauuggcuuugaaugcacgugcccagcaggcuuccagcuccuggaccagaagaccuguggcgacauugaugagugcaaggacccagaugccugcagccagaucugugucaauuacaagggcuauuuuaagugugagugcuacccuggcuacgagauggaccuacugaccaagaacugcaaggcugcugcuggcaagagcccaucccuaaucuucaccaaccggcacgaggugcggaggaucgaccuggugaagcggaacuauucacgccucauccccaugcucaagaaugucguggcacuagauguggaaguugccaccaaucgcaucuacuggugugaccucuccuaccguaagaucuauagcgccuacauggacaaggccagugacccgaaagagcaggagguccucauugacgagcaguugcacucuccagagggccuggcaguggacuggguccacaagcacaucuacuggacugacucgggcaauaagaccaucucaguggccacaguugaugguggccgccgacgcacucucuucagccguaaccucagugaaccccgggccaucgcuguugacccccugcgaggguucauguauuggucugacuggggggaccaggccaagauugagaaaucugggcucaacgguguggaccggcaaacacuggugucagacaauauugaauggcccaacggaaucacccuggaucugcugagccagcgcuuguacuggguagacuccaagcuacaccaacuguccagcauugacuucaguggaggcaacagaaagacgcugaucuccuccacugacuuccugagccacccuuuugggauagcuguguuugaggacaagguguucuggacagaccuggagaacgaggccauuuucagugcaaaucggcucaauggccuggaaaucuccauccuggcugagaaccucaacaacccacaugacauugucaucuuccaugagcugaagcagccaagagcuccagaugccugugagcugaguguccagccuaauggaggcugugaauaccugugccuuccugcuccucagaucuccagccacucucccaaguacacaugugccuguccugacacaauguggcuggguccagacaugaagaggugcuaccgagcaccucaaucuaccucaacuacgacguuagcuucuaccaugacgaggacaguaccugccaccacaagagcccccgggaccaccguccacagauccaccuaccagaaccacagcacagagacaccaagccugacagcugcagucccaagcucaguuaguguccccagggcucccagcaucagcccgucuacccuaagcccugcaaccagcaaccacucccagcacuaugcaaaugaagacaguaagaugggcucaacagucacugccgcuguuaucgggaucaucgugcccauaguggugauagcccuccugugcaugaguggauaccugaucuggagaaacuggaagcggaagaacaccaaaagcaugaauuuugacaacccagucuacaggaaaacaacagaagaagaagacgaagaugagcuccauauagggagaacugcucagauuggccaugucuauccugcagcaaucagcagcuuugaucgcccacugugggcagagcccugucuuggggagaccagagaaccggaagacccagccccugcccucaaggagcuuuuugucuugccgggggaaccaaggucacagcugcaccaacucccgaagaacccucuuuccgagcugccugucgucaaauccaagcgaguggcauuaagccuugaagaugauggacuacccugaggaugggaucacccccuucgugccucauggaauucagucccaugcacuacacucuggaugguguaugacuggaugaauggguuucuauauaugggucugugugaguguaugugugugugugauuuuuuuuuuaaauuuauguugcggaaagguaaccacaaaguuaugaugaacugcaaacauccaaaggaugugagaguuuuucuauguauaauguuuuauacacuuuuuaacugguugcacuacccaugaggaauucguggaauggcuacugcugacuaacaugaugcacauaaccaaaugggggccaauggcacaguaccuuacucaucauuuaaaaacuauauuuacagaagauguuugguugcugggggggcuuuuuuagguuuuggggcauuuguuuuuuguaaauaagaugauuaugcuuuguggcuauccaucaacauaaguaaaaaaaaaaaaaaaacacuucaacucccucccccauuuagauuauuuauuaacauauuuuaaaaaucagaugaguucuauaaauaauuuagagaagugagaguauuuauuuuuggcauguuuggcccaccacacagacucuguguguguauguguguguuuauauguguaugugugugacagaaaaaucuguagagaagaggcacaucuauggcuacuguucaaauacauaaagauaaauuuauuuucacacaguccacaagggguauaucuuguaguuuucagaaaagccuuuggaaaucuggaucagaaaauagauaccaugguuugugcaauuauguaguaaaaaaggcaaaucuuuucaccucuggcuauuccugagaccccaggaagucaggaaaagccuuucagcucacccauggcugcugugacuccuaccagggcuuucuuggcuuuggcgaaggucaguguacagacauuccaugguaccagagugcucagaaacucaagauaggauaugccucacccucagcuacuccuuguuuuaaaguucagcucuuugaguaacuucuucaauuucuuucaggacacuuggguugaauucaguaaguuuccucugaagcacccugaagggugccauccuuacagagcuaaguggagacguuuccagaucagcccaaguuuacuauagagacuggcccaggcacugaaugucuaggacaugcuguggaugaagauaaagaugguggaauagguuuuaucacaucucuuauuucucuuuuccccuuacucucuaccauuuccuuuauguggggaaacauuuuaagguaauaaauagguuacuuaccaucauauguucauauagaugaaacuaauuuuuggcuuaagucagaacaacuggccaaaauugaagucauauuugaggggggaaauggcauacgcaauauuauauuauauuggauauuuauguucacacaggaauuugguuuacugcuuuguaaauaaaaggaaaaacuccggguauauguauagauguucuucauuauagacaucuucuuugcuuuucuuggccuugggggaggaagggagaagugcucuuuucuacuuguggggucucccauuggaaacauaauccuauagucccagaaggauucaguccccaguggcuuucccauccaaagagaaagaguuugaguuucuuaacucugcuguucugccacuuacucccacuagacaaccagggacaaggugcaacauggaaguguuugacuuaaguaggagcagaggagcugcaucuaaucucaucauaccuggaacuugacacacuuaagcaaaugccuucccaucccuaccugccagaugcccccaacucaaugaaguuggaugucucaccagcuugauacccuuugaauuuucagucagacauucuggaguucuagcauccuguaccuaggaccuuccucugugucacucuuggccuccuaaacucuaagaaaauaacuauauucuggagcuugggcaguguguuuugcauaauccagcaaucuccucaugacaugcauguguugauaguccugaaacauucauugagaggguaaaugcaguugaccuagaaugaccaauaccaaacagaauuuuaagaacagguggccaacuccuauggagcuuacucacauauuacuauucuuuuaagaacggaaaguaaaauuauuuuugacugaagaaaaaugaugacagugaaaaacauggaaauguacucaaaacaagugacuuuuucuguaaccuuccaaagaaacugaauuuuccaaggaauuaaaugauaacaguggcuaaggcauaguuucuaaacuuucaguaagauccuggcauucacagaaaaaaaugaugaauggggucuggacauacagccugagaucucaaaaugacaaugaaauucacaacuuuuucucagagacauucauguuuccugcauaugcuacaacugcaguuugaaagaggcagcaaugggagcaacccuuuacaagaaacaaauugugauauauucauguguuggacggcaguaaauaagaugaaaccugaggagucagauccaccuucccccauucauagaggcuuuucagccucauuuugagguacaguuacauaucuuuugccuuuugcccccgugcauagcuaucuacagccaaucacagaucacagagucacuggacuauagagcuggaaggaagcucagagacaaugccaagggggcagaaaauuuaucagaagccagucccagugcguuuccuccauuuccuucugcaggaagacuauuuugggcugccugaacauuguaucaaaccugcuaccuauacuauggucuaccuuuccuccaguggaauuacaaaggcacuaacugaaaugccuucuagaaacagagaaaacgaaacuguacuuauuuacucuugauacacagauuauuuauaaaacagauugaaguaaccuguuaacuggcaaaaagagaaugagaucggauuuaaauguauggcaguaaguccuauugaucccuccaguuaucucaguaugacugcaguauauucauucacuaaaaccacucacuagauaccaacuacacaccuggcacugcagauguaaaggucagucacacauguucugacuuuacagaguucacaguagcaguggaggaugauauauguggaaacaaaaaaggcauugauucuauucagagcacuguuagggcucaaaggagagaggggucuuuccaccuaagaaaugaggaauagggucaucauagaagugaccuuaagucuuaaaaauuaagaaggggauuccaagcugcuucagacagagacacaucgagcuaaaacacagagguaugaaagagcacagggacuuuaggaauugcacaguucauucuaacaggaacaaaaggcucaaggggggcaagaaaugaggcuguauggaaagagauucaauguaagcacuuuauaaaauagauuaauuucugauucaaugaagcauuucuugaucauuguguacaaggcacuacaugcaucauggaaaauucauuaggaugcauugccagcacuuugcagaacugauauuauucagccucaagcuuuccaguggccaaagggaaaugcugacugcuuuucauauauuugagucaaagauuuuuuauauggucaaugaagacuaauauaagggcagugggauuuucacagaugcaugccauguugucgagagccucuuagauuuucucaacugugagaaagaaaaacgaaaauguugaagacguugagucuggagaggggauacuaaucacuguccaguugggcacuggugggaauggggaaauggcacaggaaugcaagccucuccacccuaccccccgaacuccagccauacacucaucguuucacaaaauauaaaugaguuagcauuaaauguuucagaguaaauaauuccuuuucccgaaaugcaugaagauagaguaacagacuucucacacuguauuuuuaggguauggagaauuuagaagguuaaagaauuacugcuucaauuuuucaguuaaaaaaaaaucaggaagcucuguucauucaggcuaugcaccaugugcacagucaagaauuagcagaaacccucugcauuuacaaacacuuugugcuauaaaaaaguaauuuuuaaaaagccacguguguguguguguauauauauauauauauauauauuuaaagccaagguuuugauacuuuuuuacaaaaacuacaagagaaaacaaauauaccuguccaaaccauauacuuuuaaaagagcauuuuuuuuuccauacaagcuguuguuaauuuggggguaaagugcugauuugcaaacuucaucaaauuguucccaaguggauucuccuuguuugucucccccuaccaaccccaaaguuaccauauuugauguaagaaucaggcauguuagaauguugugucacacuaacugauucugcucuuuuugucuugucauucaaguuccguuagcuucuguacgcggugcccuuugcagucuggugucucuuccagaggcgagggggcugaggauggggugcugcaucucacuagcuauacuggcaucaucuugguaaacugaaaaccaaauguggacauuuguaaaaucagugcacuguuucuagagagagauuaaauucauuuaaaaaaaa LRP8 isoform 1- Amino acid sequence(SEQ ID NO: 12) mglpepgplr llalllllll llllqlqhla aaaadpllggqgpakdcekd qfqcrnerci psvwrcdedd dcldhsdeddcpkktcadsd ftcdnghcih erwkcdgeee cpdgsdeseatctkqvcpae klscgptshk cvpaswrcdg ekdceggadeagcaticaph efqcgnrscl aavfvcdgdd dcgdgsdergcadpacgpre frcggdggga ciperwvcdr qfdcedrsdeaaelcgrpgp gatsapaaca tasqfacrsg ecvhlgwrcdgdrdckdksd eadcplgtcr gdefqcgdgt cvlaikhcnqeqdcpdgsde agclqglnec lhnnggcshi ctdlkigfectcpagfqlld qktcgdidec kdpdacsqic vnykgyfkcecypgyemdll tknckaaagk spsliftnrh evrridlvkrnysrlipmlk nvvaldveva tnriywcdls yrkiysaymdkasdpkeqev lideqlhspe glavdwvhkh iywtdsgnktisvatvdggr rrtlfsrnls epraiavdpl rgfmywsdwgdqakieksgl ngvdrqtivs dniewpngit ldllsqrlywvdsklhqlss idfsggnrkt lisstdflsh pfgiavfedkvfwtdlenea ifsanringl eisilaenln nphdivifhelkqprapdac elsvqpnggc eylclpapqi sshspkytcacpdtmwlgpd mkrcyrapqs tstttlastm trtvpattrapgttvhrsty qnhstetpsl taavpssysv prapsispstlspatsnhsq hyanedskmg stvtaavigi ivpivviallcmsgyliwrn wkrkntksmn fdnpvyrktt eeededelhigrtaqighvy paaissfdrp lwaepclget repedpapalkelfvlpgep rsqlhqlpkn plselpvvks krvalsledd gipLRP8 isoform 2- RNA sequence (SEQ ID NO: 13)gcuggcggcggccgcccagggccggggccgcgcgcccagccugagcccgccccgccgccgagcgucaccgaaccugcuugaaaugcagccgaggagccggggcgggcggcagcggcggcggcggcggcggcgggggcagcggcaaccccggcgccgcggcaaggacucggagggcugagacgcggcggcggcggcgcggggagcgcggggcgcggcggccggagccccgggcccgccaugggccuccccgagccgggcccucuccggcuucuggcgcugcugcugcugcugcugcugcugcugcugcugcagcuccagcaucuugcggcggcagcggcugauccgcugcucggcggccaagggccggccaaggauugcgaaaaggaccaauuccagugccggaacgagcgcugcauccccucuguguggagaugcgacgaggacgaugacugcuuagaccacagcgacgaggacgacugccccaagaagaccugugcagacagugacuucaccugugacaacggccacugcauccacgaacgguggaagugugacggcgaggaggaguguccugauggcuccgaugaguccgaggccacuugcaccaagcagguguguccugcagagaagcugagcuguggacccaccagccacaaguguguaccugccucguggcgcugcgacggggagaaggacugcgaggguggagcggaugaggccggcugugcuaccuggcugaacgagugucugcacaacaauggcggcugcucacacaucugcacugaccucaagauuggcuuugaaugcacgugcccagcaggcuuccagcuccuggaccagaagaccuguggcgacauugaugagugcaaggacccagaugccugcagccagaucugugucaauuacaagggcuauuuuaagugugagugcuacccuggcuacgagauggaccuacugaccaagaacugcaaggcugcugcuggcaagagcccaucccuaaucuucaccaaccggcacgaggugcggaggaucgaccuggugaagcggaacuauucacgccucauccccaugcucaagaaugucguggcacuagauguggaaguugccaccaaucgcaucuacuggugugaccucuccuaccguaagaucuauagcgccuacauggacaaggccagugacccgaaagagcaggagguccucauugacgagcaguugcacucuccagagggccuggcaguggacuggguccacaagcacaucuacuggacugacucgggcaauaagaccaucucaguggccacaguugaugguggccgccgacgcacucucuucagccguaaccucagugaaccccgggccaucgcuguugacccccugcgaggguucauguauuggucugacuggggggaccaggccaagauugagaaaucugggcucaacgguguggaccggcaaacacuggugucagacaauauugaauggcccaacggaaucacccuggaucugcugagccagcgcuuguacuggguagacuccaagcuacaccaacuguccagcauugacuucaguggaggcaacagaaagacgcugaucuccuccacugacuuccugagccacccuuuugggauagcuguguuugaggacaagguguucuggacagaccuggagaacgaggccauuuucagugcaaaucggcucaauggccuggaaaucuccauccuggcugagaaccucaacaacccacaugacauugucaucuuccaugagcugaagcagccaagagcuccagaugccugugagcugaguguccagccuaauggaggcugugaauaccugugccuuccugcuccucagaucuccagccacucucccaaguacacaugugccuguccugacacaauguggcuggguccagacaugaagaggugcuaccgagcaccucaaucuaccucaacuacgacguuagcuucuaccaugacgaggacaguaccugccaccacaagagcccccgggaccaccguccacagauccaccuaccagaaccacagcacagagacaccaagccugacagcugcagucccaagcucaguuaguguccccagggcucccagcaucagcccgucuacccuaagcccugcaaccagcaaccacucccagcacuaugcaaaugaagacaguaagaugggcucaacagucacugccgcuguuaucgggaucaucgugcccauaguggugauagcccuccugugcaugaguggauaccugaucuggagaaacuggaagcggaagaacaccaaaagcaugaauuuugacaacccagucuacaggaaaacaacagaagaagaagacgaagaugagcuccauauagggagaacugcucagauuggccaugucuauccugcagcaaucagcagcuuugaucgcccacugugggcagagcccugucuuggggagaccagagaaccggaagacccagccccugcccucaaggagcuuuuugucuugccgggggaaccaaggucacagcugcaccaacucccgaagaacccucuuuccgagcugccugucgucaaauccaagcgaguggcauuaagccuugaagaugauggacuacccugaggaugggaucacccccuucgugccucauggaauucagucccaugcacuacacucuggaugguguaugacuggaugaauggguuucuauauaugggucugugugaguguaugugugugugugauuuuuuuuuuaaauuuauguugcggaaagguaaccacaaaguuaugaugaacugcaaacauccaaaggaugugagaguuuuucuauguauaauguuuuauacacuuuuuaacugguugcacuacccaugaggaauucguggaauggcuacugcugacuaacaugaugcacauaaccaaaugggggccaauggcacaguaccuuacucaucauuuaaaaacuauauuuacagaagauguuugguugcugggggggcuuuuuuagguuuuggggcauuuguuuuuuguaaauaagaugauuaugcuuuguggcuauccaucaacauaaguaaaaaaaaaaaaaaaacacuucaacucccucccccauuuagauuauuuauuaacauauuuuaaaaaucagaugaguucuauaaauaauuuagagaagugagaguauuuauuuuuggcauguuuggcccaccacacagacucuguguguguauguguguguuuauauguguaugugugugacagaaaaaucuguagagaagaggcacaucuauggcuacuguucaaauacauaaagauaaauuuauuuucacacaguccacaagggguauaucuuguaguuuucagaaaagccuuuggaaaucuggaucagaaaauagauaccaugguuugugcaauuauguaguaaaaaaggcaaaucuuuucaccucuggcuauuccugagaccccaggaagucaggaaaagccuuucagcucacccauggcugcugugacuccuaccagggcuuucuuggcuuuggcgaaggucaguguacagacauuccaugguaccagagugcucagaaacucaagauaggauaugccucacccucagcuacuccuuguuuuaaaguucagcucuuugaguaacuucuucaauuucuuucaggacacuuggguugaauucaguaaguuuccucugaagcacccugaagggugccauccuuacagagcuaaguggagacguuuccagaucagcccaaguuuacuauagagacuggcccaggcacugaaugucuaggacaugcuguggaugaagauaaagaugguggaauagguuuuaucacaucucuuauuucucuuuuccccuuacucucuaccauuuccuuuauguggggaaacauuuuaagguaauaaauagguuacuuaccaucauauguucauauagaugaaacuaauuuuuggcuuaagucagaacaacuggccaaaauugaagucauauuugaggggggaaauggcauacgcaauauuauauuauauuggauauuuauguucacacaggaauuugguuuacugcuuuguaaauaaaaggaaaaacuccggguauauguauagauguucuucauuauagacaucuucuuugcuuuucuuggccuugggggaggaagggagaagugcucuuuucuacuuguggggucucccauuggaaacauaauccuauagucccagaaggauucaguccccaguggcuuucccauccaaagagaaagaguuugaguuucuuaacucugcuguucugccacuuacucccacuagacaaccagggacaaggugcaacauggaaguguuugacuuaaguaggagcagaggagcugcaucuaaucucaucauaccuggaacuugacacacuuaagcaaaugccuucccaucccuaccugccagaugcccccaacucaaugaaguuggaugucucaccagcuugauacccuuugaauuuucagucagacauucuggaguucuagcauccuguaccuaggaccuuccucugugucacucuuggccuccuaaacucuaagaaaauaacuauauucuggagcuugggcaguguguuuugcauaauccagcaaucuccucaugacaugcauguguugauaguccugaaacauucauugagaggguaaaugcaguugaccuagaaugaccaauaccaaacagaauuuuaagaacagguggccaacuccuauggagcuuacucacauauuacuauucuuuuaagaacggaaaguaaaauuauuuuugacugaagaaaaaugaugacagugaaaaacauggaaauguacucaaaacaagugacuuuuucuguaaccuuccaaagaaacugaauuuuccaaggaauuaaaugauaacaguggcuaaggcauaguuucuaaacuuucaguaagauccuggcauucacagaaaaaaaugaugaauggggucuggacauacagccugagauc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LRP8 isoform 2- Amino acid sequence(SEQ ID NO: 14) mglpepgplr llalllllll llllqlqhla aaaadpllggqgpakdcekd qfqcrnerci psywrcdedd dcldhsdeddcpkktcadsd ftcdnghcih erwkcdgeee cpdgsdeseatctkqvcpae klscgptshk cvpaswrcdg ekdceggadeagcatwlnec lhnnggcshi ctdlkigfec tcpagfqlldqktcgdidec kdpdacsqic vnykgyfkce cypgyemdlltknckaaagk spsliftnrh evrridlvkr nysrlipmlknvvaldveva tnriywcdls yrkiysaymd kasdpkeqevlideqlhspe glavdwvhkh iywtdsgnkt isvatvdggrrrtlfsrnls epraiavdpl rgfmywsdwg dqakieksglngvdrqtivs dniewpngit ldllsqrlyw vdsklhqlssidfsggnrkt lisstdflsh pfgiavfedk vfwtdleneaifsanringl eisilaenln nphdivifhe lkqprapdacelsvqpnggc eylclpapqi sshspkytca cpdtmwlgpdmkrcyrapqs tstttlastm trtvpattra pgttvhrstyqnhstetpsl taavpssysy prapsispst lspatsnhsqhyanedskmg stvtaavigi ivpivviall cmsgyliwrnwkrkntksmn fdnpvyrktt eeededelhi grtaqighvypaaissfdrp lwaepclget repedpapal kelfylpgeprsqlhqlpkn plselpvvks krvalsledd glp LRP8 isoform 3- RNA sequence(SEQ ID NO: 15) gcuggcggcggccgcccagggccggggccgcgcgcccagccugagcccgccccgccgccgagcgucaccgaaccugcuugaaaugcagccgaggagccggggcgggcggcagcggcggcggcggcggcggcgggggcagcggcaaccccggcgccgcggcaaggacucggagggcugagacgcggcggcggcggcgcggggagcgcggggcgcggcggccggagccccgggcccgccaugggccuccccgagccgggcccucuccggcuucuggcgcugcugcugcugcugcugcugcugcugcugcugcagcuccagcaucuugcggcggcagcggcugauccgcugcucggcggccaagggccggccaaggauugcgaaaaggaccaauuccagugccggaacgagcgcugcauccccucuguguggagaugcgacgaggacgaugacugcuuagaccacagcgacgaggacgacugccccaagaagaccugugcagacagugacuucaccugugacaacggccacugcauccacgaacgguggaagugugacggcgaggaggaguguccugauggcuccgaugaguccgaggccacuugcaccaagcagguguguccugcagagaagcugagcuguggacccaccagccacaaguguguaccugccucguggcgcugcgacggggagaaggacugcgaggguggagcggaugaggccggcugugcuaccucacugggcaccugccguggggacgaguuccaguguggggaugggacauguguccuugcaaucaagcacugcaaccaggagcaggacuguccagaugggagugaugaagcuggcugccuacaggggcugaacgagugucugcacaacaauggcggcugcucacacaucugcacugaccucaagauuggcuuugaaugcacgugcccagcaggcuuccagcuccuggaccagaagaccuguggcgacauugaugagugcaaggacccagaugccugcagccagaucugugucaauuacaagggcuauuuuaagugugagugcuacccuggcuacgagauggaccuacugaccaagaacugcaaggcugcugcuggcaagagcccaucccuaaucuucaccaaccggcacgaggugcggaggaucgaccuggugaagcggaacuauucacgccucauccccaugcucaagaaugucguggcacuagauguggaaguugccaccaaucgcaucuacuggugugaccucuccuaccguaagaucuauagcgccuacauggacaaggccagugacccgaaagagcaggagguccucauugacgagcaguugcacucuccagagggccuggcaguggacuggguccacaagcacaucuacuggacugacucgggcaauaagaccaucucaguggccacaguugaugguggccgccgacgcacucucuucagccguaaccucagugaaccccgggccaucgcuguugacccccugcgaggguucauguauuggucugacuggggggaccaggccaagauugagaaaucugggcucaacgguguggaccggcaaacacuggugucagacaauauugaauggcccaacggaaucacccuggaucugcugagccagcgcuuguacuggguagacuccaagcuacaccaacuguccagcauugacuucaguggaggcaacagaaagacgcugaucuccuccacugacuuccugagccacccuuuugggauagcuguguuugaggacaagguguucuggacagaccuggagaacgaggccauuuucagugcaaaucggcucaauggccuggaaaucuccauccuggcugagaaccucaacaacccacaugacauugucaucuuccaugagcugaagcagccaagagcuccagaugccugugagcugaguguccagccuaauggaggcugugaauaccugugccuuccugcuccucagaucuccagccacucucccaaguacacaugugccuguccugacacaauguggcuggguccagacaugaagaggugcuaccgagaugcaaaugaagacaguaagaugggcucaacagucacugccgcuguuaucgggaucaucgugcccauaguggugauagcccuccugugcaugaguggauaccugaucuggagaaacuggaagcggaagaacaccaaaagcaugaauuuugacaacccagucuacaggaaaacaacagaagaagaagacgaagaugagcuccauauagggagaacugcucagauuggccaugucuauccugcacgaguggcauuaagccuugaagaugauggacuacccugaggaugggaucacccccuucgugccucauggaauucagucccaugcacuacacucuggaugguguaugacuggaugaauggguuucuauauaugggucugugugaguguaugugugugugugauuuuuuuuuuaaauuuauguugcggaaagguaaccacaaaguuaugaugaacugcaaacauccaaaggaugugagaguuuuucuauguauaauguuuuauacacuuuuuaacugguugcacuacccaugaggaauucguggaauggcuacugcugacuaacaugaugcacauaaccaaaugggggccaauggcacaguaccuuacucaucauuuaaaaacuauauuuacagaagauguuugguugcugggggggcuuuuuuagguuuuggggcauuuguuuuuuguaaauaagaugauuaugcuuuguggcuauccaucaacauaaguaaaaaaaaaaaaaaaacacuucaacucccucccccauuuagauuauuuauuaacauauuuuaaaaaucagaugaguucuauaaauaauuuagagaagugagaguauuuauuuuuggcauguuuggcccaccacacagacucuguguguguauguguguguuuauauguguaugugugugacagaaaaaucuguagagaagaggcacaucuauggcuacuguucaaauacauaaagauaaauuuauuuucacacaguccacaagggguauaucuuguaguuuucagaaaagccuuuggaaaucuggaucagaaaauagauaccaugguuugugcaauuauguaguaaaaaaggcaaaucuuuucaccucuggcuauuccugagaccccaggaagucaggaaaagccuuucagcucacccauggcugcugugacuccuaccagggcuuucuuggcuuuggcgaaggucaguguacagacauuccaugguaccagagugcucagaaacucaagauaggauaugccucacccucagcuacuccuuguuuuaaaguucagcucuuugaguaacuucuucaauuucuuucaggacacuuggguugaauucaguaaguuuccucugaagcacccugaagggugccauccuuacagagcuaaguggagacguuuccagaucagcccaaguuuacuauagagacuggcccaggcacugaaugucuaggacaugcuguggaugaagauaaagaugguggaauagguuuuaucacaucucuuauuucucuuuuccccuuacucucuaccauuuccuuuauguggggaaacauuuuaagguaauaaauagguuacuuaccaucauauguucauauagaugaaacuaauuuuuggcuuaagucagaacaacuggccaaaauugaagucauauuugaggggggaaauggcauacgcaauauuauauuauauuggauauuuauguucacacaggaauuugguuuacugcuuuguaaauaaaaggaaaaacuccggguauauguauagauguucuucauuauagacaucuucuuugcuuuucuuggccuugggggaggaagggagaagugcucuuuucuacuuguggggucucccauuggaaacauaauccuauagucccagaaggauucaguccccaguggcuuucccauccaaagagaaagaguuugaguuucuuaacucugcuguucugccacuuacucccacuagacaaccagggacaaggugcaacauggaaguguuugacuuaaguaggagcagaggagcugcaucuaaucucaucauaccuggaacuugacacacuuaagcaaaugccuucccaucccuaccugccagaugcccccaacucaaugaaguuggaugucucaccagcuugauacccuuugaauuuucagucagacauucuggaguucuagcauccuguaccuaggaccuuccucugugucacucuuggccuccuaaacucuaagaaaauaacuauauucuggagcuugggcaguguguuuugcauaauccagcaaucuccucaugacaugcauguguugauaguccugaaacauucauugagaggguaaaugcaguugaccuagaaugaccaauaccaaacagaauuuuaagaacagguggccaacuccuauggagcuuacucacauauuacuauucuuuuaagaacggaaaguaaaauuauuuuugacugaagaaaaaugaugacagugaaaaacauggaaauguacucaaaacaagugacuuuuucuguaaccuuccaaagaaacugaauuuuccaaggaauuaaaugauaacaguggcuaaggcauaguuucuaaacuuucaguaagauccuggcauucacagaaaaaaaugaugaauggggucuggacauacagccugagaucucaaaaugacaaugaaauucacaacuuuuucucagagacauucauguuuccugcauaugcuacaacugcaguuugaaagaggcagcaaugggagcaacccuuuacaagaaacaaauugugauauauucauguguuggacggcaguaaauaagaugaaaccugaggagucagauccaccuucccccauucauagaggcuuuucagccucauuuugagguacaguuacauaucuuuugccuuuugcccccgugcauagcuaucuacagccaaucacagaucacagagucacuggacuauagagcuggaaggaagcucagagacaaugccaagggggcagaaaauuuaucagaagccagucccagugcguuuccuccauuuccuucugcaggaagacuauuuugggcugccugaacauuguaucaaaccugcuaccuauacuauggucuaccuuuccuccaguggaauuacaaaggcacuaacugaaaugccuucuagaaacagagaaaacgaaacuguacuuauuuacucuugauacacagauuauuuauaaaacagauugaaguaaccuguuaacuggcaaaaagagaaugagaucggauuuaaauguauggcaguaaguccuauugaucccuccaguuaucucaguaugacugcaguauauucauucacuaaaaccacucacuagauaccaacuacacaccuggcacugcagauguaaaggucagucacacauguucugacuuuacagaguucacaguagcaguggaggaugauauauguggaaacaaaaaaggcauugauucuauucagagcacuguuagggcucaaaggagagaggggucuuuccaccuaagaaaugaggaauagggucaucauagaagugaccuuaagucuuaaaaauuaagaaggggauuccaagcugcuucagacagagacacaucgagcuaaaacacagagguaugaaagagcacagggacuuuaggaauugcacaguucauucuaacaggaacaaaaggcucaaggggggcaagaaaugaggcuguauggaaagagauucaauguaagcacuuuauaaaauagauuaauuucugauucaaugaagcauuucuugaucauuguguacaaggcacuacaugcaucauggaaaauucauuaggaugcauugccagcacuuugcagaacugauauuauucagccucaagcuuuccaguggccaaagggaaaugcugacugcuuuucauauauuugagucaaagauuuuuuauauggucaaugaagacuaauauaagggcagugggauuuucacagaugcaugccauguugucgagagccucuuagauuuucucaacugugagaaagaaaaacgaaaauguugaagacguugagucuggagaggggauacuaaucacuguccaguugggcacuggugggaauggggaaauggcacaggaaugcaagccucuccacccuaccccccgaacuccagccauacacucaucguuucacaaaauauaaaugaguuagcauuaaauguuucagaguaaauaauuccuuuucccgaaaugcaugaagauagaguaacagacuucucacacuguauuuuuaggguauggagaauuuagaagguuaaagaauuacugcuucaauuuuucaguuaaaaaaaaaucaggaagcucuguucauucaggcuaugcaccaugugcacagucaagaauuagcagaaacccucugcauuuacaaacacuuugugcuauaaaaaaguaauuuuuaaaaagccacguguguguguguguauauauauauauauauauauauuuaaagccaagguuuugauacuuuuuuacaaaaacuacaagagaaaacaaauauaccuguccaaaccauauacuuuuaaaagagcauuuuuuuuuccauacaagcuguuguuaauuuggggguaaagugcugauuugcaaacuucaucaaauuguucccaaguggauucuccuuguuugucucccccuaccaaccccaaaguuaccauauuugauguaagaaucaggcauguuagaauguugugucacacuaacugauucugcucuuuuugucuugucauucaaguuccguuagcuucuguacgcggugcccuuugcagucuggugucucuuccagaggcgagggggcugaggauggggugcugcaucucacuagcuauacuggcaucaucuugguaaacugaaaaccaaauguggacauuuguaaaaucagugcacuguuucuagagagagauuaaauucauuuaaaaaaaaLRP8 isoform 3- Amino acid sequence (SEQ ID NO: 16)mglpepgplr llalllllll llllqlqhla aaaadpllggqgpakdcekd qfqcrnerci psvwrcdedd dcldhsdeddcpkktcadsd ftcdnghcih erwkcdgeee cpdgsdeseatctkqvcpae klscgptshk cvpaswrcdg ekdceggadeagcatslgtc rgdefqcgdg tcvlaikhcn qeqdcpdgsdeagclqglne clhnnggcsh ictdlkigfe ctcpagfqlldqktcgdide ckdpdacsqi cvnykgyfkc ecypgyemdlltknckaaag kspsliftnr hevrridlvk rnysrlipmlknvvaldvev atnriywcdl syrkiysaym dkasdpkeqevlideqlhsp eglavdwvhk hiywtdsgnk tisvatvdggrrrtlfsrnl sepraiavdp lrgfmywsdw gdqakieksglngvdrqtiv sdniewpngi tldllsqrly wvdsklhqlssidfsggnrk tlisstdfls hpfgiavfed kvfwtdleneaifsanring leisilaenl nnphdivifh elkqprapdacelsvqpngg ceylclpapq isshspkytc acpdtmwlgpdmkrcyrdan edskmgstvt aavigiivpi vviallcmsgyliwrnwkrk ntksmnfdnp vyrktteeed edelhigrta qighvypary alsleddglpLRP8 isoform 4- RNA sequence (SEQ ID NO: 17)gcuggcggcggccgcccagggccggggccgcgcgcccagccugagcccgccccgccgccgagcgucaccgaaccugcuugaaaugcagccgaggagccggggcgggcggcagcggcggcggcggcggcggcgggggcagcggcaaccccggcgccgcggcaaggacucggagggcugagacgcggcggcggcggcgcggggagcgcggggcgcggcggccggagccccgggcccgccaugggccuccccgagccgggcccucuccggcuucuggcgcugcugcugcugcugcugcugcugcugcugcugcagcuccagcaucuugcggcggcagcggcugauccgcugcucggcggccaagggccggccaaggauugcgaaaaggaccaauuccagugccggaacgagcgcugcauccccucuguguggagaugcgacgaggacgaugacugcuuagaccacagcgacgaggacgacugccccaagaagaccugugcagacagugacuucaccugugacaacggccacugcauccacgaacgguggaagugugacggcgaggaggaguguccugauggcuccgaugaguccgaggccacuugcaccaagcagguguguccugcagagaagcugagcuguggacccaccagccacaaguguguaccugccucguggcgcugcgacggggagaaggacugcgaggguggagcggaugaggccggcugugcuaccuugugcgccccgcacgaguuccagugcggcaaccgcucgugccuggccgccguguucgugugcgacggcgacgacgacuguggugacggcagcgaugagcgcggcugugcagacccggccugcgggccccgcgaguuccgcugcggcggcgauggcggcggcgccugcaucccggagcgcugggucugcgaccgccaguuugacugcgaggaccgcucggacgaggcagccgagcucugcggccguccgggccccggggccacguccgcgcccgccgccugcgccaccgccucccaguucgccugccgcagcggcgagugcgugcaccugggcuggcgcugcgacggcgaccgcgacugcaaagacaaaucggacgaggccgacugcccacugggcaccugccguggggacgaguuccaguguggggaugggacauguguccuugcaaucaagcacugcaaccaggagcaggacuguccagaugggagugaugaagcuggcugccuacaggggcugaacgagugucugcacaacaauggcggcugcucacacaucugcacugaccucaagauuggcuuugaaugcacgugcccagcaggcuuccagcuccuggaccagaagaccuguggcgacauugaugagugcaaggacccagaugccugcagccagaucugugucaauuacaagggcuauuuuaagugugagugcuacccuggcuacgagauggaccuacugaccaagaacugcaaggcugcugcuggcaagagcccaucccuaaucuucaccaaccggcacgaggugcggaggaucgaccuggugaagcggaacuauucacgccucauccccaugcucaagaaugucguggcacuagauguggaaguugccaccaaucgcaucuacuggugugaccucuccuaccguaagaucuauagcgccuacauggacaaggccagugacccgaaagagcaggagguccucauugacgagcaguugcacucuccagagggccuggcaguggacuggguccacaagcacaucuacuggacugacucgggcaauaagaccaucucaguggccacaguugaugguggccgccgacgcacucucuucagccguaaccucagugaaccccgggccaucgcuguugacccccugcgaggguucauguauuggucugacuggggggaccaggccaagauugagaaaucugggcucaacgguguggaccggcaaacacuggugucagacaauauugaauggcccaacggaaucacccuggaucugcugagccagcgcuuguacuggguagacuccaagcuacaccaacuguccagcauugacuucaguggaggcaacagaaagacgcugaucuccuccacugacuuccugagccacccuuuugggauagcuguguuugaggacaagguguucuggacagaccuggagaacgaggccauuuucagugcaaaucggcucaauggccuggaaaucuccauccuggcugagaaccucaacaacccacaugacauugucaucuuccaugagcugaagcagccaagagcuccagaugccugugagcugaguguccagccuaauggaggcugugaauaccugugccuuccugcuccucagaucuccagccacucucccaaguacacaugugccuguccugacacaauguggcuggguccagacaugaagaggugcuaccgagcaccucaaucuaccucaacuacgacguuagcuucuaccaugacgaggacaguaccugccaccacaagagcccccgggaccaccguccacagauccaccuaccagaaccacagcacagagacaccaagccugacagcugcagucccaagcucaguuaguguccccagggcucccagcaucagcccgucuacccuaagcccugcaaccagcaaccacucccagcacuaugcaaaugaagacaguaagaugggcucaacagucacugccgcuguuaucgggaucaucgugcccauaguggugauagcccuccugugcaugaguggauaccugaucuggagaaacuggaagcggaagaacaccaaaagcaugaauuuugacaacccagucuacaggaaaacaacagaagaagaagacgaagaugagcuccauauagggagaacugcucagauuggccaugucuauccugcacgaguggcauuaagccuugaagaugauggacuacccugaggaugggaucacccccuucgugccucauggaauucagucccaugcacuacacucuggaugguguaugacuggaugaauggguuucuauauaugggucugugugaguguaugugugugugugauuuuuuuuuuaaauuuauguugcggaaagguaaccacaaaguuaugaugaacugcaaacauccaaaggaugugagaguuuuucuauguauaauguuuuauacacuuuuuaacugguugcacuacccaugaggaauucguggaauggcuacugcugacuaacaugaugcacauaaccaaaugggggccaauggcacaguaccuuacucaucauuuaaaaacuauauuuacagaagauguuugguugcugggggggcuuuuuuagguuuuggggcauuuguuuuuuguaaauaagaugauuaugcuuuguggcuauccaucaacauaaguaaaaaaaaaaaaaaaacacuucaacucccucccccauuuagauuauuuauuaacauauuuuaaaaaucagaugaguucuauaaauaauuuagagaagugagaguauuuauuuuuggcauguuuggcccaccacacagacucuguguguguauguguguguuuauauguguaugugugugacagaaaaaucuguagagaagaggcacaucuauggcuacuguucaaauacauaaagauaaauuuauuuucacacaguccacaagggguauaucuuguaguuuucagaaaagccuuuggaaaucuggaucagaaaauagauaccaugguuugugcaauuauguaguaaaaaaggcaaaucuuuucaccucuggcuauuccugagaccccaggaagucaggaaaagccuuucagcucacccauggcugcugugacuccuaccagggcuuucuuggcuuuggcgaaggucaguguacagacauuccaugguaccagagugcucagaaacucaagauaggauaugccucacccucagcuacuccuuguuuuaaaguucagcucuuugaguaacuucuucaauuucuuucaggacacuuggguugaauucaguaaguuuccucugaagcacccugaagggugccauccuuacagagcuaaguggagacguuuccagaucagcccaaguuuacuauagagacuggcccaggcacugaaugucuaggacaugcuguggaugaagauaaagaugguggaauagguuuuaucacaucucuuauuucucuuuuccccuuacucucuaccauuuccuuuauguggggaaacauuuuaagguaauaaauagguuacuuaccaucauauguucauauagaugaaacuaauuuuuggcuuaagucagaacaacuggccaaaauugaagucauauuugaggggggaaauggcauacgcaauauuauauuauauuggauauuuauguucacacaggaauuugguuuacugcuuuguaaauaaaaggaaaaacuccggguauauguauagauguucuucauuauagacaucuucuuugcuuuucuuggccuugggggaggaagggagaagugcucuuuucuacuuguggggucucccauuggaaacauaauccuauagucccagaaggauucaguccccaguggcuuucccauccaaagagaaagaguuugaguuucuuaacucugcuguucugccacuuacucccacuagacaaccagggacaaggugcaacauggaaguguuugacuuaaguaggagcagaggagcugcaucuaaucucaucauaccuggaacuugacacacuuaagcaaaugccuucccaucccuaccugccagaugcccccaacucaaugaaguuggaugucucaccagcuugauacccuuugaauuuucagucagacauucuggaguucuagcauccuguaccuaggaccuuccucugugucacucuuggccuccuaaacucuaagaaaauaacuauauucuggagcuugggcaguguguuuugcauaauccagcaaucuccucaugacaugcauguguugauaguccugaaacau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aaaaaaLRP8 isoform 4- Amino acid sequence (SEQ ID NO: 18)mglpepgplr llalllllll llllqlqhla aaaadpllggqgpakdcekd qfqcrnerci psvwrcdedd dcldhsdeddcpkktcadsd ftcdnghcih erwkcdgeee cpdgsdeseatctkqvcpae klscgptshk cvpaswrcdg ekdceggadeagcaticaph efqcgnrscl aavfvcdgdd dcgdgsdergcadpacgpre frcggdggga ciperwvcdr qfdcedrsdeaaelcgrpgp gatsapaaca tasqfacrsg ecvhlgwrcdgdrdckdksd eadcplgtcr gdefqcgdgt cvlaikhcnqeqdcpdgsde agclqglnec lhnnggcshi ctdlkigfectcpagfqlld qktcgdidec kdpdacsqic vnykgyfkcecypgyemdll tknckaaagk spsliftnrh evrridlvkrnysrlipmlk nvvaldveva tnriywcdls yrkiysaymdkasdpkeqev lideqlhspe glavdwvhkh iywtdsgnktisvatvdggr rrtlfsrnls epraiavdpl rgfmywsdwgdqakieksgl ngvdrqtivs dniewpngit ldllsqrlywvdsklhqlss idfsggnrkt lisstdflsh pfgiavfedkvfwtdlenea ifsanringl eisilaenln nphdivifhelkqprapdac elsvqpnggc eylclpapqi sshspkytcacpdtmwlgpd mkrcyrapqs tstttlastm trtvpattrapgttvhrsty qnhstetpsl taavpssysv prapsispstlspatsnhsq hyanedskmg stvtaavigi ivpivviallcmsgyliwrn wkrkntksmn fdnpvyrktt eeededelhi grtaqighvy parvalsled dglpCTGF- RNA sequence (SEQ ID NO: 19)aaacucacacaacaacucuuccccgcugagaggagacagccagugcgacuccacccuccagcucgacggcagccgccccggccgacagccccgagacgacagcccggcgcgucccgguccccaccuccgaccaccgccagcgcuccaggccccgccgcuccccgcucgccgccaccgcgcccuccgcuccgcccgcagugccaaccaugaccgccgccaguaugggccccguccgcgucgccuucgugguccuccucgcccucugcagccggccggccgucggccagaacugcagcgggccgugccggugcccggacgagccggcgccgcgcugcccggcgggcgugagccucgugcuggacggcugcggcugcugccgcgucugcgccaagcagcugggcgagcugugcaccgagcgcgaccccugcgacccgcacaagggccucuucugugacuucggcuccccggccaaccgcaagaucggcgugugcaccgccaaagauggugcucccugcaucuucggugguacgguguaccgcagcggagaguccuuccagagcagcugcaaguaccagugcacgugccuggacggggcggugggcugcaugccccugugcagcauggacguucgucugcccagcccugacugccccuucccgaggagggucaagcugcccgggaaaugcugcgaggagugggugugugacgagcccaaggaccaaaccgugguugggccugcccucgcggcuuaccgacuggaagacacguuuggcccagacccaacuaugauuagagccaacugccugguccagaccacagaguggagcgccuguuccaagaccugugggaugggcaucuccacccggguuaccaaugacaacgccuccugcaggcuagagaagcagagccgccugugcauggucaggccuugcgaagcugaccuggaagagaacauuaagaagggcaaaaagugcauccguacucccaaaaucuccaagccuaucaaguuugagcuuucuggcugcaccagcaugaagacauaccgagcuaaauucuguggaguauguaccgacggccgaugcugcaccccccacagaaccaccacccugccgguggaguucaagugcccugacggcgaggucaugaagaagaacaugauguucaucaagaccugugccugccauuacaacugucccggagacaaugacaucuuugaaucgcuguacuacaggaagauguacggagacauggcaugaagccagagagugagagacauuaacucauuagacuggaacuugaacugauucacaucucauuuuuccguaaaaaugauuucaguagcacaaguuauuuaaaucuguuuuucuaacugggggaaaagauucccacccaauucaaaacauugugccaugucaaacaaauagucuaucaaccccagacacugguuugaagaauguuaagacuugacaguggaacuacauuaguacacagcaccagaauguauauuaagguguggcuuuaggagcagugggaggguaccagcagaaagguuaguaucaucagauagcaucuuauacgaguaauaugccugcuauuugaaguguaauugagaaggaaaauuuuagcgugcucacugaccugccuguagccccagugacagcuaggaugugcauucuccagccaucaagagacugagucaaguuguuccuuaagucagaacagcagacucagcucugacauucugauucgaaugacacuguucaggaaucggaauccugucgauuagacuggacagcuuguggcaagugaauuugccuguaacaagccagauuuuuuaaaauuuauauuguaaauauuguguguguguguguguguguauauauauauauauguacaguuaucuaaguuaauuuaaaguuguuugugccuuuuuauuuuuguuuuuaaugcuuugauauuucaauguuagccucaauuucugaacaccauagguagaauguaaagcuugucugaucguucaaagcaugaaauggauacuuauauggaaauucugcucagauagaaugacaguccgucaaaacagauuguuugcaaaggggaggcaucaguguccuuggcaggcugauuucuagguaggaaaugugguagccucacuuuuaaugaacaaauggccuuuauuaaaaacugagugacucuauauagcugaucaguuuuuucaccuggaagcauuuguuucuacuuugauaugacuguuuuucggacaguuuauuuguugagagugugaccaaaaguuacauguuugcaccuuucuaguugaaaauaaaguguauauuuuuucuauaaaaaaaaa aaaaaaaaCTGF- Amino Acid sequence (SEQ ID NO: 20)mtaasmgpvr vafvvllalc srpavgqncs gpercpdepaprcpagvslv ldgcgccrvc akqlgelcte rdpcdphkglfcdfgspanr kigvctakdg apcifggtvy rsgesfqssckyqctcldga vgcmplcsmd vrlpspdcpf prrvklpgkcceewvcdepk dqtvvgpala ayrledtfgp dptmiranclvqttewsacs ktcgmgistr vtndnascrl ekqsrlcmvrpceadleeni kkgkkcirtp kiskpikfel sgctsmktyrakfcgvctdg rcctphrttt lpvefkcpdg evmkknmmfiktcachyncp gdndifesly yrkmygdma LXR-a isoform 1: RNA sequence(SEQ ID NO: 21) aggaaggagggguggccugaccccucggcagucccuccccucagccuuuccccaaauugcuacuucucuggggcuccagguccugcuugugcucagcuccagcucacuggcuggccaccgagacuucuggacaggaaacugcaccauccucuucucccagcaagggggcuccagagacugcccacccaggaagucugguggccuggggauuuggacagugccuugguaaugaccagggcuccaggaagagauguccuuguggcugggggccccugugccugacauuccuccugacucugcgguggagcuguggaagccaggcgcacaggaugcaagcagccaggcccagggaggcagcagcugcauccucagagaggaagccaggaugccccacucugcuggggguacugcagggguggggcuggaggcugcagagcccacagcccugcucaccagggcagagcccccuucagaacccacagagauccguccacaaaagcggaaaaaggggccagcccccaaaaugcuggggaacgagcuaugcagcguguguggggacaaggccucgggcuuccacuacaauguucugagcugcgagggcugcaagggauucuuccgccgcagcgucaucaagggagcgcacuacaucugccacaguggcggccacugccccauggacaccuacaugcgucgcaagugccaggagugucggcuucgcaaaugccgucaggcuggcaugcgggaggaguguguccugucagaagaacagauccgccugaagaaacugaagcggcaagaggaggaacaggcucaugccacauccuugccccccagggcuuccucacccccccaaauccugccccagcucagcccggaacaacugggcaugaucgagaagcucgucgcugcccagcaacaguguaaccggcgcuccuuuucugaccggcuucgagucacgccuuggcccauggcaccagauccccauagccgggaggcccgucagcagcgcuuugcccacuucacugagcuggccaucgucucugugcaggagauaguugacuuugcuaaacagcuacccggcuuccugcagcucagccgggaggaccagauugcccugcugaagaccucugcgaucgaggugaugcuucuggagacaucucggagguacaacccugggagugagaguaucaccuuccucaaggauuucaguuauaaccgggaagacuuugccaaagcagggcugcaaguggaauucaucaaccccaucuucgaguucuccagggccaugaaugagcugcaacucaaugaugccgaguuugccuugcucauugcuaucagcaucuucucugcagaccggcccaacgugcaggaccagcuccagguagagaggcugcagcacacauauguggaagcccugcaugccuacgucuccauccaccauccccaugaccgacugauguucccacggaugcuaaugaaacuggugagccuccggacccugagcagcguccacucagagcaaguguuugcacugcgucugcaggacaaaaagcucccaccgcugcucucugagaucugggaugugcacgaaugacuguucuguccccauauuuucuguuuucuuggccggauggcugaggccugguggcugccuccuagaaguggaacagacugagaagggcaaacauuccugggagcugggcaaggagauccucccguggcauuaaaagagagucaaaggguugcgaguuuuguggcuacugagcaguggagcccucgcuaacacugugcugugucugaagaucaugcugaccccacaaacggaugggccugggggccacuuugcacaggguucuccagagcccugcccauccugccuccaccacuuccuguuuuucccacagggccccaagaaaaauucuccacugucaaaaaaaaaLXR-a (NR1H3) isoform 1: Amino acid sequence (SEQ ID NO: 22)mslwlgapvp dippdsavel wkpgaqdass qaqggsscilreearmphsa ggtagvglea aeptalltra eppsepteirpqkrkkgpap kmlgnelcsv cgdkasgfhy nvlscegckgffrrsvikga hyichsgghc pmdtymrrkc qecrlrkcrqagmreecvls eeqirlkklk rqeeeqahat slpprassppqilpqlspeq lgmieklvaa qqqcnrrsfs drirvtpwpmapdphsrear qqrfahftel aivsvqeivd fakqlpgflqlsredqiall ktsaievmll etsrrynpgs esitflkdfsynredfakag lqvefinpif efsramnelq lndaefalliaisifsadrp nvqdqlqver lqhtyvealh ayvsihhphdrlmfprmlmk lvslrtlssv hseqvfalrl qdkklpplls eiwdvheLXR-a (NR1H3) isoform 2: RNA sequence (SEQ ID NO: 23)aggaaggagggguggccugaccccucggcagucccuccccucagccuuuccccaaauugcuacuucucuggggcuccagguccugcuugugcucagcuccagcucacuggcuggccaccgagacuucuggacaggaaacugcaccauccucuucucccagcaagggggcuccagagacugcccacccaggaagucugguggccuggggauuuggacagugccuugguaaugaccagggcuccaggaagagauguccuuguggcugggggccccugugccugacauuccuccugacucugcgguggagcuguggaagccaggcgcacaggaugcaagcagccaggcccagggaggcagcagcugcauccucagagaggaagccaggaugccccacucugcuggggguacugcagggguggggcuggaggcugcagagcccacagcccugcucaccagggcagagcccccuucagaacccacagagauccguccacaaaagcggaaaaaggggccagcccccaaaaugcuggggaacgagcuaugcagcguguguggggacaaggccucgggcuuccacuacaauguucugagcugcgagggcugcaagggauucuuccgccgcagcgucaucaagggagcgcacuacaucugccacaguggcggccacugccccauggacaccuacaugcgucgcaagugccaggagugucggcuucgcaaaugccgucaggcuggcaugcgggaggaguguguccugucagaagaacagauccgccugaagaaacugaagcggcaagaggaggaacaggcucaugccacauccuugccccccagggcuuccucacccccccaaauccugccccagcucagcccggaacaacugggcaugaucgagaagcucgucgcugcccagcaacaguguaaccggcgcuccuuuucugaccggcuucgagucacggugaugcuucuggagacaucucggagguacaacccugggagugagaguaucaccuuccucaaggauuucaguuauaaccgggaagacuuugccaaagcagggcugcaaguggaauucaucaaccccaucuucgaguucuccagggccaugaaugagcugcaacucaaugaugccgaguuugccuugcucauugcuaucagcaucuucucugcagaccggcccaacgugcaggaccagcuccagguagagaggcugcagcacacauauguggaagcccugcaugccuacgucuccauccaccauccccaugaccgacugauguucccacggaugcuaaugaaacuggugagccuccggacccugagcagcguccacucagagcaaguguuugcacugcgucugcaggacaaaaagcucccaccgcugcucucugagaucugggaugugcacgaaugacuguucuguccccauauuuucuguuuucuuggccggauggcugaggccugguggcugccuccuagaaguggaacagacugagaagggcaaacauuccugggagcugggcaaggagauccucccguggcauuaaaagagagucaaaggguugcgaguuuuguggcuacugagcaguggagcccucgcuaacacugugcugugucugaagaucaugcugaccccacaaacggaugggccugggggccacuuugcacaggguucuccagagcccugcccauccugccuccaccacuuccuguuuuucccacagggccccaagaaaaauucuccacuguc aaaaaaaaaLXR-a (NR1H3) isoform 2: Amino acid sequence (SEQ ID NO: 24)mslwlgapvp dippdsavel wkpgaqdass qaqggsscilreearmphsa ggtagvglea aeptalltra eppsepteirpqkrkkgpap kmlgnelcsv cgdkasgfhy nvlscegckgffrrsvikga hyichsgghc pmdtymrrkc qecrlrkcrqagmreecvls eeqirlkklk rqeeeqahat slpprassppqilpqlspeq lgmieklvaa qqqcnrrsfs drlrvtvmlletsrrynpgs esitflkdfs ynredfakag lqvefinpifefsramnelq lndaefalli aisifsadrp nvqdqlqverlqhtyvealh ayvsihhphd rlmfprmlmk lvslrtlssvhseqvfalrl qdkklpplls eiwdvhe LXR-a (NR1H3) isoform 3: RNA sequence(SEQ ID NO: 25) aucuuacuuagggaccugcuggggugcggggaaaaggcgcagucucggugggauugcgugcaggagggucguggucuggcuguggcggaggagcauaagaagacucugcgguggagcuguggaagccaggcgcacaggaugcaagcagccaggcccagggaggcagcagcugcauccucagagaggaagccaggaugccccacucugcuggggguacugcagggguggggcuggaggcugcagagcccacagcccugcucaccagggcagagcccccuucagaacccacagagauccguccacaaaagcggaaaaaggggccagcccccaaaaugcuggggaacgagcuaugcagcguguguggggacaaggccucgggcuuccacuacaauguucugagcugcgagggcugcaagggauucuuccgccgcagcgucaucaagggagcgcacuacaucugccacaguggcggccacugccccauggacaccuacaugcgucgcaagugccaggagugucggcuucgcaaaugccgucaggcuggcaugcgggaggaguguguccugucagaagaacagauccgccugaagaaacugaagcggcaagaggaggaacaggcucaugccacauccuugccccccagggcuuccucacccccccaaauccugccccagcucagcccggaacaacugggcaugaucgagaagcucgucgcugcccagcaacaguguaaccggcgcuccuuuucugaccggcuucgagucacgccuuggcccauggcaccagauccccauagccgggaggcccgucagcagcgcuuugcccacuucacugagcuggccaucgucucugugcaggagauaguugacuuugcuaaacagcuacccggcuuccugcagcucagccgggaggaccagauugcccugcugaagaccucugcgaucgaggugaugcuucuggagacaucucggagguacaacccugggagugagaguaucaccuuccucaaggauuucaguuauaaccgggaagacuuugccaaagcagggcugcaaguggaauucaucaaccccaucuucgaguucuccagggccaugaaugagcugcaacucaaugaugccgaguuugccuugcucauugcuaucagcaucuucucugcagaccggcccaacgugcaggaccagcuccagguagagaggcugcagcacacauauguggaagcccugcaugccuacgucuccauccaccauccccaugaccgacugauguucccacggaugcuaaugaaacuggugagccuccggacccugagcagcguccacucagagcaaguguuugcacugcgucugcaggacaaaaagcucccaccgcugcucucugagaucugggaugugcacgaaugacuguucuguccccauauuuucuguuuucuuggccggauggcugaggccugguggcugccuccuagaaguggaacagacugagaagggcaaacauuccugggagcugggcaaggagauccucccguggcauuaaaagagagucaaaggguugcgaguuuuguggcuacugagcaguggagcccucgcuaacacugugcugugucugaagaucaugcugaccccacaaacggaugggccugggggccacuuugcacaggguucuccagagcccugcccauccugccuccaccacuuccuguuuuucccacagggccccaagaaaaauucuccacugucaaaaaaaaaLXR-a (NR1H3) isoform 3: Amino acid sequence (SEQ ID NO: 26)mphsaggtag vgleaaepta lltraeppse pteirpqkrkkgpapkmlgn elcsvcgdka sgfhynvlsc egckgffrrsvikgahyich sgghcpmdty mrrkcqecrl rkcniagmreecvlseeqir lkklkrqeee qahatslppr assppqilpqlspeqlgmie klvaaqqqcn rrsfsdrlry tpwpmapdphsrearqqrfa hftelaivsv qeivdfakql pgfiqlsredqiallktsai evmlletsrr ynpgsesitf lkdfsynredfakaglqvef inpifefsra mnelqlndae falliaisifsadrpnvqdq lqverlqhty vealhayvsi hhphdrlmfprmlmklvslr tlssvhseqv falrlqdkkl ppllseiwdv heLXR-a (NR1H3) isoform 4: RNA sequence (SEQ ID NO: 27)gauucuaacuuagcuaagcaaugcuacuggagaccauaggcaaagccaagguacagcuucagggaagucuuuggugagcccaucucucauuaccaagguaacgaagcgcagacuccgggcccgggugggcggcaucaccaccagguucacgccgagaaggagcuggaggagagccgcccggcuccagccggaccgcuugcccgccaucaccguuguaaucuaugcagcaaacaagcuggaacccgcuggguggcaccugcaagcagccgcccggacgcacccacucugegguggagcuguggaagccaggcgcacaggaugcaagcagccaggcccagggaggcagcagcugcauccucagagaggaagccaggaugccccacucugcuggggguacugcagggguggggcuggaggcugcagagcccacagcccugcucaccagggcagagcccccuucagaacccacagagauccguccacaaaagcggaaaaaggggccagcccccaaaaugcuggggaacgagcuaugcagcguguguggggacaaggccucgggcuuccacuacaauguucugagcugcgagggcugcaagggauucuuccgccgcagcgucaucaagggagcgcacuacaucugccacaguggeggccacugccccauggacaccuacaugcgucgcaagugccaggagugucggcuucgcaaaugccgucaggcuggcaugcgggaggaguguguccugucagaagaacagauccgccugaagaaacugaagcggcaagaggaggaacaggcucaugccacauccuugccccccagggcuuccucacccccccaaauccugccccagcucagcccggaacaacugggcaugaucgagaagcucgucgcugcccagcaacaguguaaccggcgcuccuuuucugaccggcuucgagucacgccuuggcccauggcaccagauccccauagccgggaggcccgucagcagcgcuuugcccacuucacugagcuggccaucgucucugugcaggagauaguugacuuugcuaaacagcuacccggcuuccugcagcucagccgggaggaccagauugcccugcugaagaccucugcgaucgaggugaugcuucuggagacaucucggagguacaacccugggagugagaguaucaccuuccucaaggauuucaguuauaaccgggaagacuuugccaaagcagggcugcaaguggaauucaucaaccccaucuucgaguucuccagggccaugaaugagcugcaacucaaugaugccgaguuugccuugcucauugcuaucagcaucuucucugcagaccggcccaacgugcaggaccagcuccagguagagaggcugcagcacacauauguggaagcccugcaugccuacgucuccauccaccauccccaugaccgacugauguucccacggaugcuaaugaaacuggugagccuccggacccugagcagcguccacucagagcaaguguuugcacugcgucugcaggacaaaaagcucccaccgcugcucucugagaucugggaugugcacgaaugacuguucuguccccauauuuucuguuuucuuggccggauggcugaggccugguggcugccuccuagaaguggaacagacugagaagggcaaacauuccugggagcugggcaaggagauccucccguggcauuaaaagagagucaaaggguugcgaguuuuguggcuacugagcaguggagcccucgcuaacacugugcugugucugaagaucaugcugaccccacaaacggaugggccugggggccacuuugcacaggguucuccagagcccugcccauccugccuccaccacuuccuguuuuucccacagggccccaagaaaaauucuccacugucaaaaaaaaaLXR-a (NR1H3) isoform 4: Amino acid sequence (SEQ ID NO: 28)mqqtswnplg gtckqppgrt hsavelwkpg aqdassqaqggsscilreea rmphsaggta gvgleaaept alltraeppsepteirpqkr kkgpapkmlg nelcsvcgdk asgfhynvlscegckgffrr svikgahyic hsgghcpmdt ymrrkcqecrlrkcrqagmr eecvlseeqi rlkklkrqee eqahatslpprassppqilp qlspeqlgmi eklvaaqqqc nrrsfsdrlrvtpwpmapdp hsrearqqrf ahftelaivs vqeivdfakqlpgflqlsre dqiallktsa ievmlletsr rynpgsesitflkdfsynre dfakaglqve finpifefsr amnelqlndaefalliaisi fsadrpnvqd qlqverlqht yvealhayvsihhphdrlmf prmlmklvsl rtlssvhseq vfalrlqdkk lppllseiwd vheLXR-b (NR1H2) isoform 1: RNA sequence (SEQ ID NO: 29)ucgucaaguuucacgcuccgccccucuuccggacgugacgcaagggcgggguugccggaagaaguggcgaaguuacuuuugaggguauuugaguagcggcggugugucaggggcuaaagaggaggacgaagaaaagcagagcaagggaacccagggcaacaggaguaguucacuccgcgagaggccguccacgagacccccgcgcgcagccaugagccccgccccccgcuguugcuuggagaggggcgggaccuggagagaggcugcuccgugaccccaccauguccucuccuaccacgaguucccuggauaccccccugccuggaaauggccccccucagccuggcgccccuucuucuucacccacuguaaaggaggaggguccggagccguggcccggggguccggacccugaugucccaggcacugaugaggccagcucagccugcagcacagacugggucaucccagaucccgaagaggaaccagagcgcaagcgaaagaagggcccagccccgaagaugcugggccacgagcuuugccgugucuguggggacaaggccuccggcuuccacuacaacgugcucagcugcgaaggcugcaagggcuucuuccggcgcagugugguccgugguggggccaggcgcuaugccugccgggguggcggaaccugccagauggacgcuuucaugcggcgcaagugccagcagugccggcugcgcaagugcaaggaggcagggaugagggagcagugcguccuuucugaagaacagauccggaagaagaagauucggaaacaacagcagcaggagucacagucacagucgcagucaccuguggggccgcagggcagcagcagcucagccucugggccuggggcuuccccugguggaucugaggcaggcagccagggcuccggggaaggcgaggguguccagcuaacagcggcucaagaacuaaugauccagcaguugguggcggcccaacugcagugcaacaaacgcuccuucuccgaccagcccaaagucacgcccuggccccugggcgcagacccccagucccgagaugcccgccagcaacgcuuugcccacuucacggagcuggccaucaucucaguccaggagaucguggacuucgcuaagcaagugccugguuuccugcagcugggccgggaggaccagaucgcccuccugaaggcauccacuaucgagaucaugcugcuagagacagccaggcgcuacaaccacgagacagaguguaucaccuucuugaaggacuucaccuacagcaaggacgacuuccaccgugcaggccugcagguggaguucaucaaccccaucuucgaguucucgcgggccaugcggcggcugggccuggacgacgcugaguacgcccugcucaucgccaucaacaucuucucggccgaccggcccaacgugcaggagccgggccgcguggaggcguugcagcagcccuacguggaggcgcugcuguccuacacgcgcaucaagaggccgcaggaccagcugcgcuucccgcgcaugcucaugaagcuggugagccugcgcacgcugagcucugugcacucggagcaggucuucgccuugcggcuccaggacaagaagcugccgccucugcugucggagaucugggacguccacgagugaggggcuggccacccagccccacagccuugccugaccacccuccagcagauagacgccggcaccccuuccucuuccuaggguggaaggggcccugggccgagccuguagaccuaucggcucucaucccuugggauaagccccaguccagguccaggaggcucccucccugcccagcgagucuuccagaaggggugaaaggguugcaggucccgaccacugacccuucccggcugcccucccuccccagcuuacaccucaagcccagcacgcagugcaccuugaacagagggaggggaggacccauggcucuccccccuagcccgggagaccagggccuuccucuuccucugcuuuuauuuaauaaaaacuaaaaacagaaacaggaaaauaaaauaugaauacaauccagcccggagcuggagugcaLXR-b (NR1H2) isoform 1: Amino acid sequence (SEQ ID NO: 30)msspttssld tplpgngppq pgapsssptv keegpepwpggpdpdvpgtd eassacstdw vipdpeeepe rkrkkgpapkmlghelcrvc gdkasgfhyn vlscegckgf frrsvvrggarryacrgggt cqmdafmrrk cqqcrlrkck eagmreqcvlseeqirkkki rkqqqqesqs qsqspvgpqg ssssasgpgaspggseagsq gsgegegvql taaqelmiqq lvaaqlqcnkrsfsdqpkvt pwplgadpqs rdarqqrfah ftelaiisvqeivdfakqvp gflqlgredq iallkastie imlletarrynhetecitfl kdftyskddf hraglqvefi npifefsramrrlglddaey alliainifs adrpnvqepg rvealqqpyveallsytrik rpqdqlrfpr mlmklvslrt lssvhseqvf alrlqdkklp pllseiwdvh eLXR-b (NR1H2) isoform 2: RNA sequence (SEQ ID NO: 31)ucgucaaguuucacgcuccgccccucuuccggacgugacgcaagggcgggguugccggaagaaguggcgaaguuacuuuugaggguauuugaguagcggcggugugucaggggcuaaagaggaggacgaagaaaagcagagcaagggaacccagggcaacaggaguaguucacuccgcgagaggccguccacgagacccccgcgcgcagccaugagccccgccccccgcuguugcuuggagaggggcgggaccuggagagaggcugcuccgugaccccaccauguccucuccuaccacgaguucccuggauaccccccugccuggaaauggccccccucagccuggcgccccuucuucuucacccacuguaaaggaggaggguccggagccguggcccggggguccggacccugaugucccaggcacugaugaggccagcucagccugcagcacagacuggggcguccuuucugaagaacagauccggaagaagaagauucggaaacaacagcagcaggagucacagucacagucgcagucaccuguggggccgcagggcagcagcagcucagccucugggccuggggcuuccccugguggaucugaggcaggcagccagggcuccggggaaggcgaggguguccagcuaacagcggcucaagaacuaaugauccagcaguugguggcggcccaacugcagugcaacaaacgcuccuucuccgaccagcccaaagucacgcccuggccccugggcgcagacccccagucccgagaugcccgccagcaacgcuuugcccacuucacggagcuggccaucaucucaguccaggagaucguggacuucgcuaagcaagugccugguuuccugcagcugggccgggaggaccagaucgcccuccugaaggcauccacuaucgagaucaugcugcuagagacagccaggcgcuacaaccacgagacagaguguaucaccuucuugaaggacuucaccuacagcaaggacgacuuccaccgugcaggccugcagguggaguucaucaaccccaucuucgaguucucgcgggccaugcggcggcugggccuggacgacgcugaguacgcccugcucaucgccaucaacaucuucucggccgaccggcccaacgugcaggagccgggccgcguggaggcguugcagcagcccuacguggaggcgcugcuguccuacacgcgcaucaagaggccgcaggaccagcugcgcuucccgcgcaugcucaugaagcuggugagccugcgcacgcugagcucugugcacucggagcaggucuucgccuugcggcuccaggacaagaagcugccgccucugcugucggagaucugggacguccacgagugaggggcuggccacccagccccacagccuugccugaccacccuccagcagauagacgccggcaccccuuccucuuccuaggguggaaggggcccugggccgagccuguagaccuaucggcucucaucccuugggauaagccccaguccagguccaggaggcucccucccugcccagcgagucuuccagaaggggugaaaggguugcaggucccgaccacugacccuucccggcugcccucccuccccagcuuacaccucaagcccagcacgcagugcaccuugaacagagggaggggaggacccauggcucuccccccuagcccgggagaccagggccuuccucuuccucugcuuuuauuuaauaaaaacuaaaaacagaaacaggaaaauaaaauaugaauacaauccagcccggagcuggagug caLXR-b (NR1H2) isoform 2: Amino acid sequence (SEQ ID NO: 32)msspttssld tplpgngppq pgapsssptv keegpepwpggpdpdvpgtd eassacstdw gvlseeqirk kkirkqqqqesqsqsqspvg pqgssssasg pgaspggsea gsqgsgegegvqltaaqelm iqqlvaaqlq cnkrsfsdqp kvtpwplgadpqsrdarqqr fahftelaii svqeivdfak qvpgflqlgredqiallkas tieimlleta rrynheteci tflkdftyskddfhraglqv efinpifefs ramrrlgldd aeyalliainifsadrpnvq epgrvealqq pyveallsyt rikrpqdqlrfprmlmklvs lrtlssvhse qvfalrlqdk klppllseiw dvhe has-miR-199a-1 sequence(SEQ ID NO: 33) GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC has-miR-199a-2 sequence (SEQ ID NO: 34)AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCA AGGGAGAGCAhas-miR-1908 sequence (SEQ ID NO: 35)CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC has-miR-7-1 sequence (SEQ ID NO: 36)UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAU GCCUCUACAGhas-miR-7-2 sequence (SEQ ID NO: 37)CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGCC AGCCAUCGCAhas-miR-7-3 sequence (SEQ ID NO: 38)AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGAC UCCCUUCGACmiR-Zip 199a-3p sequence (SEQ ID NO: 39)GATCCGACAGTAGCCTGCACATTAGTCACTTCCTGTCAGTAACCAATGTG CAGACTACTGTTTTTTGAATTmiR-Zip 199a-5p sequence (SEQ ID NO: 40)GATCCGCCCAGTGCTCAGACTACCCGTGCCTTCCTGTCAGGAACAGGTAGTCTGAACACTGGGTTTTTGAATT miR-Zip 1908 sequence (SEQ ID NO: 41)GATCCGCGGCGGGAACGGCGATCGGCCCTTCCTGTCAGGACCAATCGCCG TCCCCGCCGTTTTTGAATTmiR-Zip 7 sequence (SEQ ID NO: 42)GATCCGTGGAAGATTAGTGAGTTTATTATCTTCCTGTCAGACAACAAAATCACTAGTCTTCCATTTTTGAATT

The members of this network can be used as targets for treatingmetastatic melanoma. In addition, the members can be used a biomarkersfor determining whether a subject has, or is at risk of having, ametastatic melanoma or for determining a prognosis or surveillance ofpatient having the disorder. Accordingly, the present inventionencompasses methods of treating metastatic melanoma by targeting one ormore of the members, methods of determining the efficacy of therapeuticregimens for inhibiting the cancer, and methods of identifyinganti-cancer agent. Also provided are methods of diagnosing whether asubject has, or is at risk for having, metastatic melanoma, and methodsof screening subjects who are thought to be at risk for developing thedisorder. The invention also encompasses various kits suitable forcarrying out the above mentioned methods.

ApoE Polypeptides

The term “polypeptide or peptide” as used herein includes recombinantlyor synthetically produced fusion or chimeric versions of any of theaforementioned metastasis suppressors, having the particular domains orportions that are involved in the network. The term also encompasses ananalog, fragment, elongation or derivative of the peptide (e.g. thathave an added amino-terminal methionine, useful for expression inprokaryotic cells).

“Apolipoprotein polypeptide or ApoE polypeptide” as used herein means apeptide, drug, or compound that mimics a function of the nativeapolipoprotein either in vivo or in vitro including apolipoproteinanalogs, fragments, elongations or derivatives that are a peptide ofbetween 10 and 200 amino acid residues in length, such peptides cancontain either natural, or non-natural amino acids containing amidebonds. Apolipoprotein peptide fragments may be modified to improve theirstability or bioavailability in vivo as known in the art and may containorganic compounds bound to the amino acid side chains through a varietyof bonds.

In one aspect, our invention is a method for using an isolated apoEp1.Bpeptide having the amino acid sequence TQQIRLQAEIFQAR (murine)(SEQ. ID.No.43) or AQQIRLQAEAFQAR (human)(SEQ. ID. No.44) or an analog, fragment,elongation or derivative of the peptide. The invention also includes anucleic acid molecule encoding the apoEpI.B peptide, or an analog,fragment, elongation or derivative thereof.

The term “analog” includes any peptide having an amino acid residuesequence substantially identical to the native peptide in which one ormore residues have been conservatively substituted with a functionallysimilar residue and which displays the ability to mimic the nativepeptide. Examples of conservative substitutions include the substitutionof one non-polar (hydrophobic) residue such as alanine, isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a nonderivatized residueprovided that such polypeptide displays the requisite activity. Analogsof the peptides include peptides having the following sequences:TAQIRLQAEIFQAR (SEQ.ID.NO.:45); TQAIRLQAEIFQAR (SEQ.ID.NO.:46);TQQARLQAEIFQAR (SEQ.ID.NO.:47) and TQQIALQAEIFQAR (SEQ.ID.NO.:48).

“Derivative” refers to a peptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included asderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

The term “fragment” refers to any subject peptide having an amino acidresidue sequence shorter than that of a peptide whose amino acid residuesequence is shown herein.

The term “elongation” refers to any subject peptide having an amino acidsequence longer by one or two amino acids (either at the carboxy oramino terminal end) than that of a peptide of the present invention.Preferably, the elongation occurs at the amino terminal end. Fragmentsand elongations of the peptides include peptides that have the followingsequences: QTQQIRLQAEIFQAR (SEQ.ID.NO.:49) and QQIRLQAEIFQAR(SEQ.ID.NO.:50).

ApoE polypeptides and methods for their preparation are described inU.S. Pat. No. 6,652,860, incorporated herein by reference.

LXR Agonists

The methods of the invention can include administering a LXR agonist forthe prevention and treatment of metastasis. The LXR agonist can be acompound according to the Formula I, II, III, or IV shown below.

Formula I is provided below:

or a pharmaceutically acceptable salt thereof, wherein

Ar is an aryl group;

R¹ is a member selected from the group consisting of

—OH, —CO₂H, —O—(C₁-C₇)alkyl, —OC(O)—, —(C₁-C₇)alkyl,—O—(C₁-C₇)heteroalkyl, —OC(O)—(C₁-C₇)heteroalkyl, —NH₂, —NH(C₁-C₇)alkyl, —N((C₁-C₇)alkyl)₂ and —NH—S(O)₂(C₁-C₅)alkyl;

R² is a member selected from the group consisting of

(C₁-C₇)alkyl, (C₁-C₇)heteroalkyl, aryl and aryl (C₁-C₇)alkyl;

X¹, X², X³, X⁴, X⁵ and X⁶ are each independently a member selected fromthe group consisting of:

H, (C¹-C⁵)alkyl, (C¹-C⁵)heteroalkyl, F and CI, with the proviso that nomore than three of X¹ through X⁶ are H, (C¹-C⁵)alkyl,(C¹-C⁵)heteroalkyl; and

Y is a divalent linking group selected from the group consisting of:—N(R¹²)S(O)_(m)—, —N(R¹²)S(O)_(m)N(R¹³)—, —N(R¹²)C(O)—,—N(R¹²)C(O)N(R¹³)—, —N(R¹²)C(S)— and —N(R¹²)C(O)O—;

wherein R¹² and R¹³ are each independently selected from the groupconsisting of:

H, (C₁-C₇)alkyl, (C₁-C₇)heteroalkyl, aryl and aryl(C₁-C₇)alkyl, andoptionally when Y is —N(R¹²)S(O)_(m)— or —N(R¹²)S(O)_(m)N(R¹³)—, R′²forms a five- or six-membered ring fused to Ar or to R² through covalentattachment to Ar or to R², respectively; and the subscript m is aninteger of from 1 to 2;

with the proviso that when R¹ is OH, and —Y—R² is —N(R¹²)S(O)_(m)—R² or—N(R¹²)C(O)N(R¹³)—R² and is attached to a position para to thequaternary carbon attached to Ar, and when R² is phenyl, benzyl, orbenzoyl, then i) at least one of R¹² or R¹³ is other than hydrogen andcontains an electron-withdrawing substituent, or ii) R² is substitutedwith a moiety other than amino, acetamido, di(C₁-C₇)alkylamino,(C₁-C₇)alkylamino, halogen, hydroxy, nitro, or (C₁-C₇)alkyl, or iii) thebenzene ring portion of R² is substituted with at least threeindependently selected groups in addition to the Y group or point ofattachment to Y.

In some embodiments, Y is —N(R12)S(O)2- and R1 is OH.

Accordingly, the compounds of Formula I include but are not limited thecompound with the structure shown below:

Compounds of Formula I can be synthesized as described by U.S. Pat. No.6,316,503, incorporated herein by reference.

Formula II is provided below:

wherein:

R¹ is —H;

X¹ is a bond, C₁ to C₅ alkyl, —C(O)—, —C(═CR⁸R⁹)—, —O—, —S(O)_(t)—,—NR⁸—, —CR⁸R⁹—, —CHR²³,

—CR⁸(CR⁹)—, —C(CR⁸)₂—, —CR₈(OC(O)R⁹)—, —C═NOR⁹—, —C(O)NR⁸—, —CH₂O—,—CH₂S—, —CH₂NR⁸—, —OCH₂—,—SCH₂—, —NR⁸CH₂—, or

R² is H, C₁ to C₆alkyl, C₂ to C₆alkenyl, C₂ to C₆alkynyl, C₃ to C₆cycloalkyl, —CH₂OH, C₇ to C₁₁ arylalkyl, phenyl, naphthyl, C₁ to C₃perfluoroalkyl, CN, C(O)NH₂, CO₂R¹² or phenyl substituted independentlyby one or more of the groups independently selected from C₁ to C₃ alkyl,C₂ to C₄ alkenyl, C₂ to C₄ alkynyl, C₁ to C₃ alkoxy, C₁ to C₃perfluoroalkyl, halogen, —NO₂, —NR⁸R⁹, —CN, —OH, and C₁ to C₃alkylsubstituted with 1 to 5 fluorines, or R² is a heterocycle selected fromthe group consisting of pyridine, thiophene, benzisoxazole,benzothiophene, oxadiazole, pyrrole, pyrazole, imidazole, and furan,each of which may be optionally substituted with one to three groupsindependently selected from C₁ to C₃alkyl, C₁ to C₃ alkoxy, C₁ to C₃perfluoroalkyl, halogen, —NO₂, —NR⁸R⁹, —CN, and C₁ to C₃alkylsubstituted with 1 to 5 fluorines;

X² is a bond or —CH₂—;

R³ is phenyl, naphthyl, or phenyl or naphthyl substituted independentlyby one to four groups independently selected from C₁ to C₃ alkyl,hydroxy, phenyl, acyl, halogen, —NH₂, —CN, —NO₂, C₁ to C₃ alkoxy, C₁ toC₃perfluoroalkyl, C₁ to C₃ alkyl substituted with 1 to 5 fluorines,NR¹⁴R¹⁵, —C(O)R¹⁰,

—C(O)NR¹⁰R¹¹, —C(O)NR¹¹A, —CH≡CHR⁸, —WA, —C≡CA, —CH═CHA, —WYA,—WYNR¹¹-A,—WYR¹⁰, —WY(CH2)_(j)A, —WCHR¹¹(CH2)_(j)A, —W(CH2)_(j)A, —W(CH2)_(j)R¹⁰,—CHR¹¹W(CH2)_(j)R¹⁰, —CHR¹¹W(CH2)_(j)A, —CHR¹¹NR¹²YA, —CHR¹¹NR¹²YR¹⁰pyrrole, —W(CH2)_(j)A(CH2)_(k)D(CH2)_(p)Z,—W(CR¹⁸R¹⁹)A(CH₂)_(k)D(CH₂)_(p)Z, —(CH₂)_(j)WA(CH₂)_(k)D(CH₂)_(p)Z,—CH═CHA(CH₂)_(k)D(CH₂)_(p)Z, —C≡CA(CH₂)_(k)D(CH₂)_(p)Z,—W(CH₂)_(j)C≡CA(CH₂)_(k)D(CH₂)_(p)Z, and —W(CH₂)_(j)Z, or R³ is aheterocycle selected from pyrimidine, thiophene, furan, benzothiophene,indole, benzofuran, benzimidazole, benzothiazole, benzoxazole, andquinoline, each of which may be optionally substituted with one to threegroups independently selected from C₁ to C₃alkyl, C₁ to C₃ alkoxy,hydroxy, phenyl, acyl, halogen, —NH₂, —CN, —NO₂, C₁ to C₃perfluoroalkyl, C₁ to C₃ alkyl substituted with 1 to 5 fluorines,—C(O)R¹⁰, —C(O)NR¹⁰R¹¹, —C(O)NR¹¹A, —C≡CR⁸, —CH═CHR⁸, —WA, —C≡CA,—CH═CHA, —WYA, —WYR¹⁰, —WY(CH2)_(j)A,—W(CH₂)_(j)A, —W(CH₂)_(j)R¹⁰, —CHR¹¹W(CH₂)_(j)R¹⁰, —CHR¹¹W(CH₂)_(j)A,—CHR¹¹NR¹²YA, —CHR¹¹NR¹²YR¹⁰,—WCHR¹¹(CH₂)_(j)A, —W(CH₂)_(j)A(CH₂)_(k)D(CH₂)_(p)Z,—W(CR¹⁸R¹⁹)A(CH₂)_(k)D(CH₂)_(p)Z, —(CH₂)_(j)WA(CH₂)_(k)D(CH₂)_(p)Z,—CH═CHA(CH₂)_(k)D(CH₂)_(p)Z, —C≡CA(CH₂)_(k)D(CH₂)_(p)Z,W(CH₂)_(j)C≡CA(CH₂)_(k)D(CH₂)_(p)Z, and —W(CH₂)_(j)Z;

W is a bond, —O—, —S—, —S(O)—, —S(O)₂—, —NR¹¹—, or —N(COR¹²)—;

Y is —CO—, —S(O)²—, —CONR¹³, —CONR¹³CO—, —CONR¹³SO₂—, —C(NCN)—, —CSNR¹³,—C(NH)NR¹³, or —C(O)O—;

j is 0 to 3;

k is 0 to 3;

t is 0 to 2;

D is a bond, —CH═CH—, —C≡C—, —C═, —C(O)—, phenyl, —O—, —NH—, —S—,—CHR¹⁴—, —CR¹⁴R¹⁵—, —OCHR¹⁴, —OCR¹⁴R¹⁵—, or —CH(OH)CH(OH)—;

p is 0 to 3;

Z is —CO₂R¹¹, —CONR¹⁰R¹¹, —C(NR¹⁰)NR¹¹R¹², —CONH₂NH², —CN, —CH₂OH,—NR¹⁶R¹⁷, phenyl, CONHCH(R²⁰)COR¹², phthalimide, pyrrolidine-2,5dione,thiazolidine-2,4-dione, tetrazolyl, pyrrole, indole, oxazole,2-thioxo-1,3-thiazolinin-4-one, C₁ to C₇ amines, C₃ to C₇ cyclic amines,or C₁ to C₃ alkyl substituted with one to two OH groups; wherein saidpyrrole is optionally substituted with one or two substituentsindependently selected from the group consisting of —CO₂CH₃, —CO₂H,—COCH₃, —CONH₂, and

—CN;

wherein said C₁ to C₇amines are optionally substituted with one to twosubstituents independently

selected from the group consisting of —OH, halogen, —OCH₃, and —C≡CH;

wherein said phenyl is optionally substituted with CO₂R¹¹, and whereinsaid C₃ to C₇ cyclic amines are optionally substituted with one or twosubstituents independently selected from the group consisting of—OH—CH₂OH, C₁ to C₃ alkyl, —CH₂OCH₃, —CO₂CH₃, and —CONH₂, and whereinsaid oxazole is optionally substituted with CH₂CO₂R¹¹;

A is phenyl, naphthyl, tetrahydronaphthyl, indan or biphenyl, each ofwhich may be optionally substituted by one to four groups independentlyselected from halogen, C₁ to C₃ alkyl, C₂ to C₄ alkenyl, C₂ to C₄alkynyl, acyl, hydroxy, halogen, —CN, —NO₂, —CO₂R¹¹, —CH₂CO₂R¹¹, phenyl,C₁ to C₃perfluoroalkoxy, C₁ to C₃ perfluoroalkyl, —NR¹⁰R¹¹, —CH₂NR¹⁰R¹¹,—SR¹¹, C₁ to C₆ alkyl substituted with 1 to 5 fluorines, C₁ to C₃alkylsubstituted with 1 to 2-OH groups, C₁ to C₆ alkoxy optionallysubstituted with 1 to 5 fluorines, or phenoxy optionally substitutedwith 1 to 2 CF₃ groups; or

A is a heterocycle selected from pyrrole, pyridine, pyridine-N-oxide,pyrimidine, pyrazole, thiophene, furan, quinoline, oxazole, thiazole,imidazole, isoxazole, indole, benzo[1,3]-dioxole,benzo[1,2,5]-oxadiazole, isochromen-1-one, benzothiophene,benzofuran,2,3-di-5 hydrobenzo[1,4]-dioxine, bitheinyl,quinazolin-2,4-9[3H]dione, and 3-H-isobenzofuran-1-one, each of whichmay be optionally substituted by one to three groups independentlyselected from halogen, C₁ to C₃ alkyl, acyl, hydroxy, —CN, —NO₂, C₁ toC₃perfluoroalkyl, —NR¹⁰R¹¹, CH₂NR¹⁰R¹¹, —SR¹¹, C₁ to C₃ alkylsubstituted with 1 to 5 fluorines, and C₁ to C₃ alkoxy optionallysubstituted with 1 to 5 fluorines;

R⁴, R⁵, and R⁶ are each, independently, —H or —F;

R⁷ is C₁ to C₄ alkyl, C₁ to C₄ perfluoroalkyl, halogen, —NO₂, —CN,phenyl or phenyl substituted with one or two groups independentlyselected from halogen, C₁ to C₂alkyl and OH;

provided that if X₁R² forms hydrogen, then R³ is selected from:

(a) phenyl substituted by —W(CH₂)_(j)A(CH₂)_(k)D(CH₂)_(p)Z,—W(CR¹⁸R¹⁹)A(CH₂)_(k)D(CH₂)_(p)Z, —(CH₂)_(j)WA(CH₂)_(k)D(CH₂)_(p)Z,—CH═CHA(CH₂)_(k)D(CH₂)_(p)Z, —C≡CA(CH₂)_(k)D(CH₂)_(p)Z, or—W(CH₂)_(j)C≡CA(CH₂)_(k)D(CH₂)_(p)Z, wherein the phenyl moiety isfurther optionally substituted with one or two groups independentlyselected from C₁ to C₂ alkyl, C₁ to C₂perfluoroalkyl, halogen, and CN;and

(b) a heterocycle selected from pyrimidine, thiophene, and furan, eachof which is substituted by one of —W(CH₂)_(j)A(CH₂)_(k)D(CH₂)_(p)Z,—W(CR¹⁸R¹⁹)A(CH₂)_(k)D(CH₂)_(p)Z, —(CH₂)_(j)WA(CH₂)_(k)D(CH₂)_(p)Z,

—CH═CHA(CH₂)_(k)D(CH₂)_(p)Z, —C≡CA(CH₂)_(k)D(CH₂)_(p)Z, or—W(CH₂)_(j)C≡CA(CH₂)_(k)D(CH₂)_(p)Z;

each R⁸ is independently-H, or C₁ to C₃alkyl;

each R⁹ is independently-H, or C₁ to C₃alkyl;

each R¹⁰ is independently-H, —CH, C₁ to C₃alkoxy, C₁ to C₇ alkyl, C₃ toC₇ alkenyl, C₃ to C₇ alkynyl, C₃ to C₇ cycloalkyl, —CH₂CH₂OCH₃,2-methyl-tetrahydro-furan, 2-methyl-tetrahydro-pyran,4-methyl-piperidine, morpholine, pyrrolidine, or phenyl optionallysubstituted with one or two C₁ to C₃alkoxy groups, wherein said C₁ to C₇alkyl is optionally substituted with 1, 2 or 3 groups independentlyselected from C₁ to C₃ alkoxy, C₁ to C₃thioalkoxy, and CN;

each R¹¹ is independently-H, C₁ to C₃alkyl or R²²; or R¹⁰ and R¹¹, whenattached to the same atom, together with said atom form:

a 5 to 7 membered saturated ring, optionally substituted by 1 to 2groups independently selected from C₁ to C₃ alkyl, OH and C₁-C₃alkoxy;or a 5 to 7 membered ring containing 1 or 2 heteroatoms,

optionally substituted by 1 to 2 groups independently selected from C₁to C₃alkyl, OH and C₁-C₃ alkoxy;

each R¹² is independently-H, or C₁ to C₃alkyl;

each R¹³ is independently-H, or C₁ to C₃alkyl;

each R¹⁴ and R¹⁵ is, independently, C₁ to C₇ alkyl, C₃ to C₈ cycloalkyl,C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, —CH, —F, C₇ to C₁₄arylalkyl, wheresaid arylalkyl is optionally substituted with 1 to 3 groupsindependently selected from NO₂, C₁ to C₆ alkyl, C₁ to C₃perhaloalkyl,halogen, CH₂CO₂R¹¹, phenyl and C₁ to C₃ alkoxy, or R¹² and R¹⁵ togetherwith the atom to which they are attached can form a 3 to 7 memberedsaturated ring;

each R¹⁶ and R¹⁷ is, independently, hydrogen, C₁ to C₃ alkyl, C₁ toC₃alkenyl, C₁ to C₃ alkynyl, phenyl, benzyl or C₃ to C₈ cycloalkyl,wherein said C₁ to C₃ alkyl is optionally substituted with one OH group,and wherein said benzyl is optionally substituted with 1 to 3 groupsselected from C₁ to C₃alkyl and C₁ to C₃alkoxy; or R¹⁶ and R¹⁷, togetherwith the atom to which they are attached, can form a 3 to 8 memberedheterocycle which is optionally substituted with one or two substituentsindependently selected from the group consisting of C₁ to C₃alkyl, —OH,CH₂OH, —CH₂OCH₃, —CO₂CH₃, and —CONH₂;

each R¹⁸ and R¹⁹ is, independently, C₁ to C₃alkyl;

each R²⁰ is independently H, phenyl, or the side chain of a naturallyoccurring alpha amino acid;

each R²² is independently arylalkyl optionally substituted with CH2COOH;and

each R₂₃ is phenyl;

or a pharmaceutically acceptable salt thereof

Compounds of Formula II can be synthesized as described in U.S. Pat. No.7,576,215, incorporated herein by reference. The compound of formula IIcan be any of compounds 26-32, or a pharmaceutically acceptable saltthereof.

Formula III is provided below:

wherein:

X is selected from hydrogen, C₁-C₈ alkyl, halo, —OR¹⁰, —NR¹⁰R¹¹, nitro,cyano, —COOR¹⁰, or —COR¹⁰.

Z is CH, CR³ or N, wherein when Z is CH or CR³, k is 0-4 and t is 0 or1, and when Z is N, k is 0-3 and t is 0;

Y is selected from —O—, —S—, —N(R¹²)—, and —C(R⁴)(R⁵)—;

W¹ is selected from C₁-C₆ alkyl, C₀-C₆ alkyl, C₃-C₆ cycloalkyl, aryl andHet, wherein said C₁-C₈ alkyl, C₃-C₈ cycloalkyl, Ar and Het areoptionally unsubstituted or substituted with one or more groupsindependently selected from halo, cyano, nitro, C₁-C₆ alkyl, C₃-C₆alkenyl, C₃-C₆ alkynyl, —C₀-C₆ alkyl-CO₂R¹², —C₀-C₆alkyl-C(O)SR¹²,—C₀-C₆alkyl-CONR¹³R¹⁴, —C₀-C₆ alkyl-COR¹⁵, —C₀-C₆ alkyl-NR¹³R¹⁴,—C₀-C₆—C₀-C₆alkyl-OR¹², —C₀-C₆alkyl-SO₃H, —C₀-C₆alkyl-SO₂NR¹³R¹⁴,—C₀-C₆alkyl-SO₂R¹², —C₀-C₆alkyl-SOR¹⁵, —C₀-C₆alkylOCOR¹⁵, —C₀-C₆alkyl-OC(O)NR¹³R¹⁴, —C₀-C₆alkyl-OC(O)OR¹⁵, —C₀-C₆ alkyl-NR¹³C(O)OR¹⁵, —C₀-C₆alkyl-NR¹³C(O)NR¹³R¹⁴ and —C₀-C₆ alkyl-NR¹³COR¹⁵, where said C₁-C₆alkyl, is optionally unsubstituted or substituted by one or more halosubstituents;

W² is selected from H, halo, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl,—C₀-C₆ alkyl-NR¹³R¹⁴, —C₀-C₆alkyl-SR¹², —C₀-C₆ alkyl-OR¹²,—C₀-C₆alkylCO₂R¹², —C₀-C₆alkyl-C(O)SR¹², —C₀-C₆ alkylCONR¹³R¹⁴,—C₀-C₆alkyl-COR¹⁵, —C₀-C₆ alkylOCOR¹⁵, —C₀-C₆alkyl-OCONR¹³R¹⁴,—Co-C6alkyl-NR¹³CONR¹³R¹⁴, —C₀-C₆ alkyl-NR¹³COR¹⁵, —C₀-C₆alkyl-Het,—C₀-C₆alkyl-Ar and —C₀-C₆alkyl-C₃-C₇ cycloalkyl, wherein said C₁-C₆alkyl is optionally unsubstituted or substituted by one or more halosubstituents, and wherein the C3-C7cycloalkyl, Ar and Het moieties ofsaid —C₀-C₆alkyl-Het, —C₀-C₆alkyl-Ar and —C₀-C₆alkyl-C3-C7cycloalkyl areoptionally unsubstituted or substituted with one or more groupsindependently selected from halo, cyano, nitro, C₁-C₆ alkyl, C₃-C₆alkenyl, C₃-C₆ alkynyl, —C₀-C₆alkyl-CO₂R¹², —C₀-C₆ alkyl-C(O)SR¹²,—C₀-C₆alkyl-CONR¹³R¹⁴, —C₀-C₆alkyl-COR¹⁵, —C₀-C₆alkyl-NR¹³R¹⁴,—C₀-C₆alkyl-SR¹², —C₀-C₆alkyl-OR′², —C₀-C₆ alkyl-SO₃H,—C₀-C₆alkyl-SO₂NR¹³R¹⁴, —C₀-C₆alkyl-SO₂R¹², —C₀-C₆alkyl-SOR¹⁵,—C₀-C₆alkyl- OCOR¹⁵, —C₀-C₆alkylOC(O)NR¹³R¹⁴, —C₀-C₆alkyl-OC(O)OR¹⁵,—C₀-C₆alkyl-NR¹³C(O)OR¹⁵, —C₀-C₆alkyl-NR¹³C(O)NR¹³R¹⁴, and—C₀-C₆alkyl-NR¹³COR¹⁵, where said C₁-C₆ alkyl, is optionallyunsubstituted or substituted by one or more halo substituents;

W³ is selected from the group consisting of: H, halo, C₁-C₆ alkyl,—C₀-C₆ alkyl-NR¹³R¹⁴, —C₀-C₆alkylSR¹², —C₀-C₆alkyl-OR¹²,—C₀-C₆alkyl-CO₂R′², —C₀-C₆alkyl-C(O)SR¹², —C₀-C₆alkyl-CONR¹³R¹⁴,

—C₀-C₆alkyl-COR¹⁵, —C₀-C₆alkyl-OCOR¹⁵, —C₀-C₆ alkyl-OCONR¹³R¹⁴,—C₀-C₆alkylNR¹³CONR¹³R¹⁴,—C₀-C₆alkyl-NR¹³COR¹⁵, —C₀-C₆alkyl-Het, —C₁-C₆alkyl-Ar and—C₁-C₆alkyl-C₃-C₇cycloalkyl, wherein said C₁-C₆ alkyl is optionallyunsubstituted or substituted by one or more halo substituents;

Q is selected from C₃-C₈cycloalkyl, Ar and Het; wherein saidC₃-C₈cycloalkyl, Ar and Het are optionally unsubstituted or substitutedwith one or more groups independently selected from halo, cyano, nitro,C₁-C₆alkyl, C₃-C₆alkenyl, C₃-C₆alkynyl, —C₀-C₆alkylCO₂R¹², —C₀-C₆alkyl-C(O)SR¹², —C₀-C₆alkylCONR¹³R¹⁴, —C₀-C₆ alkyl-COR¹⁵,—C₀-C₆alkylNR¹³R¹⁴, —C₀-C₆alkyl-SR¹², —C₀-C₆alkyl-OR¹², —C₀-C₆alkyl-SO₃H, —C₀-C₆ alkyl-SO₂NR¹³R¹⁴, —C₀-C₆alkyl-SO₂R¹²,—C₀-C₆alkyl-SOR¹⁵, —C₀-C₆alkyl-OCOR¹⁵,

—C₀-C₆alkyl-OC(O)NR¹³R¹⁴, —C₀-C₆alkyl-OC(O)OR¹⁵,—C₀-C₆alkylNR¹³C(O)OR¹⁵, —C₀-C₆ alkyl-NR¹³C(O)NR¹³R¹⁴, and—C₀-C₆alkyl-NR¹³COR¹⁵, where said C₁-C₆alkyl is optionally unsubstitutedor substituted by one or more halo substituents;

p is 0-8;

n is 2-8;

m is 0 or 1;

q is 0 or 1;

t is 0 or 1;

each R¹ and R² are independently selected from H, halo, C₁-C₆alkyl,C₃-C₆alkenyl, C₃-C₆ alkynyl, —C₀-C₆alkyl-NR¹³R¹⁴, —C₀-C₆alkyl-OR¹²,—C₀-C₆ alkyl-SR¹², —C₁-C₆alkyl-Het, —C₁-C₆alkyl-Ar and—C₁-C₆alkyl-C₃-C₇cycloalkyl, or R¹ and R² together with the carbon towhich they are attached form a 3-5 membered carbocyclic or heterocyclicring, wherein said heterocyclic ring contains one, or more heteroatomsselected from N, O, and S, where any of said C₁-C₆ alkyl is optionallyunsubstituted or substituted by one or more halo substituents;

each R³ is the same or different and is independently selected fromhalo, cyano, nitro, C₁-C₆ alkyl, C₃-C₆alkenyl, C₃-C₆alkynyl,—C₀-C₆alkyl-Ar, —C₀-C₆alkyl-Het, —C₀-C₆alkyl-C₃-C₇cycloalkyl,—C₀-C₆alkyl-CO₂R¹², —C₀-C₆alkyl-C(O)SR¹², —C₀-C₆alkyl-CONR¹³R¹⁴,—C₀-C₆alkyl-COR¹⁵, —C₀-C₆alkyl-NR¹³R¹⁴, —C₀-C₆alkyl-SR¹²,—C₀-C₆alkyl-OR¹², —C₀-C₆alkyl-SO₃H, —C₀-C₆alkylSO₂NR¹³R¹⁴, —C₀-C₆alkyl-SO₂R², —C₀-C₆alkylSOR¹⁵, —C₀-C₆alkyl-OCOR¹⁵, —C₀-C₆alkyl-OC(O)NR¹³R¹⁴, —C₀-C₆alkyl-OC(O)OR¹⁵,

—C₀-C₆alkyl-NR¹³C(O)OR¹⁵, —C₀-C₆alkyl-NR¹³C(O)NR¹³R¹⁴, and—C₀-C₆alkyl-NR¹³COR¹⁵, wherein said C₁-C₆alkyl is optionallyunsubstituted or substituted by one or more halo substituents;

each R⁴ and R⁵ is independently selected from H, halo, C₁-C₆alkyl,—C₀-C₆alkyl-Het, —C₀-C₆alkyl-Ar and —C₀-C₆alkyl-C₃-C₇cycloalkyl;

R⁶ and R⁷ are each independently selected from H, halo, C₁-C₆ alkyl,—C₀-C₆alkyl-Het, —C₀-C₆ alkyl-Ar and —C₀-C₆alkyl-C₃-C₇cycloalkyl;

R⁸ and R⁹ are each independently selected from H, halo, C₁-C₆ alkyl,—C₀-C₆alkyl-Het, —C₀-C₆ alkyl-Ar and —C₀-C₆alkyl-C₃-C₇ cycloalkyl;

R¹⁰ and R¹¹ are each independently selected from H, C₁-C₁₂ alkyl,C₃-C₁₂alkenyl, C₃-C₁₂alkynyl,

—C₀-C₈alkyl-Ar, —C₀-C₈ alkyl-Het, —C₀-C₈ alkyl-C₃-C₇ cycloalkyl, —C₀-C₈alkyl-O—Ar, —C₀-C₈ alkyl-O-Het,—C₀-C₈ alkyl-O—C₃-C₇cycloalkyl, —C₀-C₈alkyl-S(O)X—C₀-C₆alkyl,—C₀-C₈alkyl-S(O)X—Ar, —C₀-C₈ alkyl-S(O)X-Het, —C₀-C₈alkyl-S(O)X—C₃-C₇cycloalkyl, —C₀-C₈alkyl-NH—Ar, —C₀-C₈alkyl-NH-Het,—C₀-C₈alkyl-NH—C3-C7cycloalkyl, —C₀-C₈alkyl-N(C₁-C₄ alkyl)-Ar,—C₀-C₈alkyl-N(C₁-C₄alkyl)-Het, —C₀-C₈alkyl-N(C₁-C₄alkyl-C3-C7cycloalkyl,—C₀-C₈alkyl-Ar, —C₀-C₈alkyl-Het and —C₀-C₈alkyl-C₃-C₇cycloalkyl, where xis 0, 1, or 2, or 10° and R¹¹, together with the nitrogen to which theyare attached, form a 4-7 membered heterocyclic ring which optionallycontains one or more additional heteroatoms selected from N, O, and S,wherein said C₁-C₁₂alkyl, C₃-C₁₂ alkenyl, or C₃-C₁₂alkynyl is optionallysubstituted by one or more of the substituents independently selectedfrom the group halo, —OH, —SH, —NH₂, —NH (unsubstituted C₁-C₆alkyl), —N(unsubstituted C₁-C₆ alkyl)(unsubstituted C₁-C₆alkyl), unsubstituted—OC₁-C₆ alkyl, —CO₂H, —CO₂(unsubstituted C₁-C₆ alkyl), —CONH₂, —CONH(unsubstituted C₁-C₆ alkyl), —CON(unsubstituted C₁-C₆alkyl)(unsubstituted C₁-C₆ alkyl), —SO₃H, —SO₂NH₂, —SO₂NH(unsubstitutedC₁-C₆alkyl) and—SO₂N(unsubstituted C₁-C₆alkyl)(unsubstituted C₁-C₆ alkyl);

R¹² is selected from H, C₁-C₆ alkyl, C3-C6alkenyl, C3-C6alkynyl,—C₀-C₆alkyl-Ar, —C₀-C₆alkyl-Het and —C₀-C₆alkyl-C3-C7cycloalkyl;

each R¹³ and each R¹⁴ are independently selected from H, C₁-C₆alkyl,C₃-C₆alkenyl, C₃-C₆alkynyl, —C₀-C₆alkyl-Ar, —C₀-C₆alkyl-Het and—C₀-C₆alkyl-C3-C7cycloalkyl, or R¹³ and R¹⁴ together with the nitrogento which they are attached form a 4-7 membered heterocyclic ring whichoptionally contains one or more additional heteroatoms selected from N,O, and S;

and R¹⁵ is selected from C₁-C₆alkyl, C₃-C₆ alkenyl, C₃-C₆alkynyl,—C₀-C₆alkyl-Ar, —C₀-C₆ alkyl-Het and —C₀-C₆ alkyl-C₃-C₇ cycloalkyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, X is hydrogen, p is 0, t is 0, Z is CH, and Y is—O—.

In further embodiments, X is hydrogen, p is 0, t is 0, Z is CH, and Y is—O—, W¹ and W² are phenyl, W³ is hydrogen, q is 1, and R⁸ and R⁹ arehydrogen.

In other embodiments, X is hydrogen, p is 0, t is 0, Z is CH, and Y is—O—, W¹ and W² are phenyl, W³ is hydrogen, q is 1, R⁸ and R⁹ arehydrogen, and Q is Ar.

Accordingly, the compounds of Formula III include but are not limitedthe compounds with structures shown below GW3965 2 and SB742881 25:

Compounds of Formula III can be synthesized as described in U.S. Pat.Nos. 7,365,085 and 7,560,586 incorporated herein by reference.

Formula IV is shown below:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   J¹¹ is —N═ and J²¹ is —CR³⁰⁰—, or J¹¹ is —CR²⁰⁰— and J21 is ═N—;    -   R⁰⁰ is G¹, G²¹ or R^(N);    -   R²⁰⁰ is G¹, G²¹, or R^(C);    -   R³⁰⁰ and R⁴⁰⁰ are independently R^(C) or Q, provided one and        only one of R³⁰⁰, R⁴⁰⁰, and    -   R⁵⁰⁰ is

Q;

-   -   Q is C₃₋₆ cycloalkyl, heteroaryl or heterocyclyl, each        optionally substituted with 1 to 4R^(Q), or Q is        —X—Y—Z; wherein each R^(Q) is independently aryloxy, aralkyloxy,        aryloxyalkyl, arylC₀-C₆alkylcarboxy, C(R¹¹⁰)═C(R¹¹⁰)—COOH, oxo,        ═S, —Z, —Y′—Z, or —X—Y—Z, wherein each R^(Q) is optionally        substituted with 1 to 4 R⁸⁰;

R⁵⁰⁰ is G¹ G²¹, Q, or R^(C); provided that only one of R⁰⁰, R²⁰⁰, andR⁵⁰⁰ is G¹ and only one of R⁰⁰, N═, and R⁵⁰⁰ is G²¹;

G²¹ is -J⁰-K⁰, wherein J⁰ and K⁰ are independently aryl or heteroaryl,each optionally substituted with one to four R^(K) groups; each R^(K) isindependently hydrogen, halogen, CR¹¹⁰═CR¹¹⁰COOR¹¹⁰, nitro, —Z, —Y—Z, or—X—Y—Z;

G¹ is -L¹⁰-R, wherein L¹⁰ is a bond, L⁵⁰, L⁶⁰, -L⁵⁰-L⁶⁰-L⁵⁰-, L or-L⁶⁰-L⁵⁰-L⁵⁰-, wherein

each L⁵⁰ is independently —[C(R¹⁵⁰)₂]_(m)—;

each L⁶⁰ is independently —CS—, —CO—, —SO₂—, —O—, —CON(R¹¹⁰)—,—CONR¹¹⁰N(R¹¹⁰)—, —C(═NR¹¹⁰)—, —C(NOR¹¹)—, —C(═N—N(R¹¹⁰)₂)—,—C₃-C₈cycloalkyl-, or -heterocyclyl-, wherein the cycloalkyl orheterocyclyl is optionally substituted with one to 4 R¹⁴⁰ groups; or oreach L⁶⁰ is independently C₂-C₆ alidiyl, wherein the alidiyl chain isoptionally interrupted by —C(R¹⁰⁰)₂—, —C(R¹¹⁰)₂C(R¹¹⁰)_(z)—,—C(R¹¹)C(R¹¹⁰)—, —C(R¹¹⁰)₂O—, —C(R¹¹⁰)_(z)NR¹¹⁰—, —CC—, —O—, —S—,—N(RO)CO—, —N(R¹⁰⁰)CO₂—, —CON(R¹¹⁰)—, —CO—, —CO₂—, —OC(═O)—,—OC(═O)N(R¹⁰⁰)—, —SO₂—, N(R¹⁰⁰)SO₂—, or

—SO₂N(R¹⁰⁰);

R is aryl, heterocyclyl, heteroaryl or —(C₃-C₆)cycloalkyl, wherein R isoptionally substituted with 1 to 4 R^(A), wherein each R^(A) isindependently halogen, nitro, heterocyclyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, (C₃-C₈ cycloalkyl)-C₁-C₆ alkyl-, (C₃-C₈cycloalkenyl)-C₁-C₆ alkyl-, (C₃-C₈ cycloalkyl)-C₁-C₆ alkenyl-,arylalkyl, aryloxy, arylC₁₋₆ alkoxy, C₁-C₆ haloalkyl, SO₂R¹¹⁰, OR¹¹⁰,SR¹¹⁰, N₃, SOR¹¹⁰, COR¹¹⁰, SO₂N(R¹¹⁰)₂, SO₂NR¹¹⁰COR¹¹⁰, C≡N, C(O)OR¹¹⁰,CON(R¹¹⁰)₂, —CON(R¹¹⁰)OR¹¹⁰, OCON(R¹¹⁰)₂, —NR¹¹⁰COR¹¹⁰, NR¹¹⁰CON(R¹¹⁰)₂,NR¹¹⁰COOR¹¹⁰, —C(═N—OH)R¹¹⁰, —C(═S)N(R¹¹⁰)₂, —S(═O)N(R¹¹⁰)₂,—S(═O)OR¹¹⁰, —N(R¹¹⁰)S(═O)₂R¹¹⁰, —C(═O)N(R¹¹⁰)N(R¹¹⁰)₂, —OC(═O)—R¹¹⁰,—OC(═O)—OR¹¹⁰ or N(R¹¹)₂, wherein each R^(A) is optionally substitutedwith 1 to 4 groups which independently are -halogen, —C₁-C₆ alkyl,aryloxy, C₀₋₆ alkylSO₂R¹¹⁰, C₀₋₆ alkylCOOR¹¹⁰ C₁₋₆ alkoxyaryl, C₁-C₆haloalkyl,

—SO₂R¹¹⁰, —OR¹¹⁰, SO₂R¹¹⁰, —COR¹¹⁰, —SO₂N(R¹¹⁰)₂, —SO₂NR¹¹⁰COR¹¹⁰, —C≡N,—C(O)OR¹¹⁰,—CON(R¹¹⁰)₂, —CON(R¹¹⁰)OR¹¹⁰, —OCON(R¹¹⁰)₂, —NR¹¹⁰CON(R¹¹⁰)₂,—NR¹¹⁰COOR¹¹⁰, or —N(R¹¹⁰)₂;

R^(N) is -L³¹-R⁶⁰, wherein L³¹ is a bond, —X³(CH_(z))_(m)—X³—,—(CH₂)_(m)—X3-(CH₂)_(n)— or —(CH₂)_(1+w), —Y³—(CH₂)_(w)—, wherein each wis independently 0-5: and each X³ is independently a bond, —C(R¹¹⁰)₂—,—C(R¹¹⁰)₂C(R¹¹⁰)₂—, —C(R¹¹⁰)═C(R¹¹⁰)—, —CO—, —CS—, —CONR¹⁰⁰—,—C(═N)(R¹⁰⁰)—, —C(═N—OR¹¹⁰—, —C[═N—N(R¹¹⁰)₂], —CO₂—, —SO₂—, or—SO₂N(R¹¹⁰)—; and

Y³ is —O—, —S—, —NR⁷⁰—, —N(R¹⁰⁰)CO—, —N(R¹¹⁰)CO2-, —OCO—,—OC(═O)N)(R¹⁰⁰)—, —NR¹⁰⁰CONR¹⁰⁰—, —N(R¹¹⁰)SO₂—, or —NR¹⁰⁰CSNR¹⁰⁰—;

or L³¹ is C₂₋₆ alidiyl chain wherein the alidiyl chain is optionallyinterrupted by) —C(R¹¹⁰)₂—, —C(R¹¹⁰)₂C(R¹¹⁰)₂—, —C(R¹¹⁰)═C(R¹¹⁰)—,—C(R¹¹⁰)₂O—, —C(R¹¹⁰)₂NR¹¹⁰—, —C≡C—, —O—, —S—, —N(R¹⁰⁰)CO—,

—N(R¹⁰⁰)CO₂—, —CON(R¹⁰⁰)—, —CO—, —CO₂—, —OC(═O)—, —OC(═O)N(R¹¹⁰)—,—SO₂—, —N(R¹⁰⁰)SO₂—, or) —SO₂N(R¹⁰⁰); and

R⁶⁰ is C₁-C₆ alkyl, C₁-C₆ halo alkyl, aryl, C₃-C₈ cycloalkyl,heteroaryl, heterocyclyl, —CN, —C(═O)R¹¹⁰, —C(═O)OR¹¹⁰, —C(═O)N(R¹¹⁰)₂,—N(R¹¹⁰)₂, —SO₂R¹¹⁰, —S(═O)₂N(R¹¹⁰)₂, —C(═O)N(R¹¹⁰)N(R¹¹⁰)₂, or—C(═O)N(R¹¹)(OR¹¹⁰), wherein the aryl, heteroaryl, cycloalkyl, orheterocyclyl is optionally substituted with 1 to 4 R^(60a), wherein

each R^(60a) is independently —Z, —Y′—Z, or —X—Y—Z;

each R^(C) is independently -L³⁰-R⁷⁰, wherein

each L³⁰ is independently a bond or —(CH₂)_(m)—V¹⁰—(CH₂)_(n)—, wherein

V¹⁰ is —C(R¹¹⁰)₂—, —C(R¹¹⁰)₂C(R¹¹⁰)₂, —C(R¹¹⁰)═C(R¹¹⁰)—, —C(R¹¹⁰)₂O—,—C(R¹¹⁰)₂NR¹¹⁰—, —C≡C—, —O—,

—S—, —NR¹⁰—, —N(R¹⁰⁰)CO—, —N(R¹⁰⁰)CO2-, —OCO—, —CO—, —CS—, —CONR¹⁰⁰)—,—C(═N—R¹¹⁰)—, —C(═N—) OR¹¹⁰)—,—C[═N—N(R¹¹⁰)₂], —CO₂—, —OC(═O)—, —OC(═O)N(R¹⁰⁰)—, SO₂—, —N(R¹⁰⁰)SO₂—,—SO₂N(R¹⁰⁰)—, —NR¹⁰⁰CONR¹⁰⁰—, —NR¹⁰⁰CSNR¹⁰⁰—, C₃-C₆cyclo alkyl, or C₃-C₆cyclohaloalkyl; or each L³⁰ is independently C₂-C₆ alidiyl, wherein thealidiyl chain is optionally interrupted by —C(R¹¹⁰)₂—,—C(R¹¹⁰)₂C(R¹¹⁰)₂—, —C(R¹¹⁰)C(R¹¹⁰)—, —C(R¹¹⁰)₂O—, —C(R¹¹⁰)₂NR¹¹⁰—,—C≡C—, —O—, —S—, —N(R¹⁰⁰)CO—, —N(R¹⁰⁰)CO₂—, —NR¹¹⁰—, —CON(R¹⁰⁰)—, —CO—,—CO₂—, —O(C═O)—, —O(C═O)N(R¹⁰⁰)—, —SO₂—, —N(R¹⁰⁰)SO₂—, or —SO₂N(R¹⁰⁰)—;

each R⁷⁰ is independently hydrogen, halogen, nitro, aryl, heteroaryl,heterocyclyl, —Z, —Y—Z, or —X—YZ,

wherein the aryl, heteroaryl, and heterocyclyl, are each optionallysubstituted with 1 to 4 R^(70a), wherein each R^(70a) is independentlyaryloxy, aralkyloxy, aryloxyalkyl, arylC₀-C₆alkylcarboxy,C(R¹¹⁰)═C(R¹¹⁰)COOH, oxo, —Z, —Y′—Z, or —X—Y—Z, wherein each R^(70a) isoptionally substituted with 1 to 4 R⁸⁰, and wherein each R⁸⁰ isindependently halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₈haloalkyl, C₁-C₈haloalkyl(OR¹¹⁰), C₀-C₆ alkylOR¹¹⁰, C₀-C₆ alkylCON(R¹¹⁰)₂, C₀-C₆alkylCOR¹¹⁰, C₀-C₆ alkylCOOR¹¹⁰, or C₀-C₆ alkylSO₂R¹¹⁰;

each R¹⁰⁰ is independently —R¹¹⁰, —C(═O)R¹¹⁰, —CO2R¹¹⁰, or —SO₂R¹¹⁰;

each R¹¹⁰ is independently -hydrogen, —C₁-C₆ alkyl, C₂-C₆ alkenyl, C₁-C₆alkynyl, —C₁-C₆ haloalkyl, or —N(R¹²)₂, wherein any of R¹¹⁰ isoptionally substituted with 1 to 4 radicals of R¹²⁰;

each R¹²⁰ is independently halogen, cyano, nitro, oxo, —B(OR¹³⁰), C₀-C₆alkylN(R¹³)₂, C₁-C₆haloalkyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, (C₀-C₆alkyl)C═O(OR¹³⁰), C₀-C₆ alkylOR¹³⁰, C₀-C₆ alkylCOR¹³⁰,

C₀-C₆alkylSO₂R¹³⁰, C₀-C₆alkylCON(R¹³)₂, C₀-C₆alkylCONR¹³⁰OR¹³⁰,C₀-C₆alkylSO₂N(R¹³⁰)₂, C₀-C₆alkylSR¹³⁰, C₀-C₆ haloalkylOR¹³⁰,C₀-C₆alkylCN, —C₀-C₆alkyN(R¹³)₂, —NR¹³SO₂R¹³, or —OC₀₋₆ alkylCOOR¹³⁰;

each R¹³⁰ is independently hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl;

each R¹⁴⁰ is independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, C₁-C₆haloalkyl, C₀-C₆) alkylCON(R¹¹⁰)_(o), C₀-C₆ alkylCONR¹¹⁰R¹⁰, C₀-C₆alkylOR¹¹⁰, or C₀-C₆ alkylCOOR¹¹⁰; and

each R¹⁵⁰ is independently hydrogen, halogen, OR¹³⁰, (C₁-C₆)alkyl or(C₁-C₆)haloalkyl, wherein

each alkyl is optionally substituted with at least one group which areeach independently halogen, cyano, nitro, azido, OR¹³⁰, C(O)R¹³⁰,C(O)OR¹³C(O)N(R¹³⁰)₂, N(R¹³⁰)₂, N(R¹³⁰)C(O)R¹³⁰, N(R¹³⁰)S(O)₂R¹³⁰,—OC(O)OR¹³⁰, OC(O)N(R¹³⁰)2, N(R¹³⁰)C(O)OR¹³⁰, N(R¹³⁰)C(O)N(R¹³⁰), SR¹³⁰,S(O)R¹³⁰, S(O)₂R′, or S(O)₂N(R¹³⁰)₂; or two R¹⁵⁰ (bonded to same ordifferent atoms) can be taken together to form a C3-C6 cycloalkyl;

each X is independently —O—, —S—, or —N(R¹⁰⁰)—;

each Y is independently —[C(R¹⁵⁰)₂]_(p)—, or —C₂-C₆ alkenyl, wherein pis 1, 2, 3, 4, 5, or 6;

each Y′ is independently —[C(R¹⁵⁰)₂]_(p)—, —C₂-C₆ alkenyl C₃-C₈cycloalkyl, or heterocyclyl, wherein the cycloalkyl or heterocyclyl isoptionally substituted with 1 to 3 Z groups;

each Z is independently —H, halogen, —OR¹¹⁰, —SR¹¹⁰, —C(═O)R¹¹⁰,—C(═O)OR¹¹⁰, —C(═O)N(R¹¹⁰)₂,

—N(R¹⁰⁰)₂, —N₃, —NO₂, —C(═N—OH)R¹¹⁰, —C(═S)N(R¹¹⁰)₂, —CN, —S(═O)R¹¹⁰,—S(═O)N(R¹¹⁰)₂, —S(═O)OR¹¹⁰,—S(═O)₂R¹¹⁰, S(═O)₂N(R¹¹⁰)₂, —NR¹¹⁰COR¹¹⁰, —N(R¹¹⁰)C(═O)N(R¹¹⁰)₂,—N(R¹¹⁰)COOR¹¹⁰, —N(R¹¹⁰)S(═O)₂R¹¹⁰, —C(═O)N(R¹¹⁰)N(R¹¹⁰)₂,—C(═O)N(R¹¹⁰)(OR¹¹⁰), —OC(═O)—R¹¹⁰, —OC(═O)—OR¹¹⁰, or—OC(═O)—N(R¹¹⁰)₂; and

each m and n is independently 0, 1, 2, 3, 4, 5, or 6.

In some embodiments the compound of Formula IV has a structure ofFormula V or VI:

In other embodiments the compound of Formula VI has a structure ofFormula VII:

In yet other embodiments the compound of Formula VI has a structure ofFormula VIII:

In still further embodiments the compound of Formula VI has a structureof Formula IX:

Accordingly, the compounds of Formula IV which can be useful in themethods of the invention include, but are not limited to, compoundshaving the structures are shown below, and pharmaceutically acceptablesalts thereof:

and selected from the list comprising:

332-(1-(3chloro-3′-fluoro-4′-(hydroxymethyl)-5′-(methylsulfonyl)biphenyl-4-yl)-2-(2-(2,6dichlorophenyl)propan-2-yl)-1H-imidazol-4-yl)propan-2-ol; 342-(2-(2(2-chloro-3-fluorophenyl)propan-2-yl)-1-(3′-fluoro-4′-(hydroxymethyl)-5′(methylsulfonyl)biphenyl-4-yl)-1H-imidazol-4-yl)propan-2-ol;352-(2-(2(2,6-dichlorophenyl)propan-2-yl)-1-(3′-fluoro-4′-(hydroxymethyl)-5′(methylsulfonyl)biphenyl-4-yl)-1H-imidazol-4-yl)propan-2-ol;362-(2-(2(2,6-dichlorophenyl)propan-2-yl)-1-(3,3′-difluoro-4′-(hydroxymethyl)-5′(methylsulfonyl)biphenyl-4-yl)-1H-imidazol-4-yl)propan-2-ol;and 372-(2-[1(2,6-dichlorophenyl)ethyl]-1-[3,3′-difluoro-4′-(hydroxymethyl)-5′(methylsulfonyl)biphenyl-4-yl]-1H-imidazol-4-yl)propan-2-ol.Compound 12 is also known as WO2010 0138598 Ex. 9. Compound 38 is alsoknown WO2007 002563 Ex. 19. Compound 39 is also known as WO2012 0135082.

Compounds of Formula IV can be synthesized as described in PCTpublication No. US2010/0069367 and WO2010/138598 incorporated herein byreference.

The LXR agonist that can be used for the treatment and/or prevention ofmetastasis can be compound 24, or a pharmaceutically acceptable saltthereof.

In further embodiments compounds that can be used for the treatmentand/or prevention of metastasis can be found in the PCT publications inthe list consisting of: WO2006/094034, WO2008/049047, WO2009/020683,WO2009/086138, WO2009/086123, WO2009/086130, WO2009/086129,WO2007/002559, WO2007/002563, WO2007/081335, WO2006/017055,WO2006/102067, WO2009/024550, US2006/0074115, US2006/0135601,WO2009/021868, WO2009/040289, WO2007/047991, WO2007/050425,WO2006/073363, WO2006/073364, WO2006/073365, WO2006/073366,WO2006/073367, US2009/0030082, WO2008/065754, JP2008/179562,WO2007/092065, US2010/0069367, U.S. Pat. No. 7,998,995, U.S. Pat. No.7,247,748, WO2010/138598, U.S. Pat. No. 7,365,085, US75776215, U.S.63/136,503, US2004/0072868, US2005/0107444, US2005/0113580,US2005/0131014, US2005/0282908, US2009/0286780, incorporated herein byreference.

LXRα and LXRβ, initially discovered by multiple groups at roughly thesame time (Apfel et al., 1994; Willy et al., 1995; Song et al., 1994;Shinar et al., 1994; Teboul et al., 1995), belong to a family of nuclearhormone receptors that are endogenously activated by cholesterol and itsoxidized derivatives to mediate transcription of genes involved inmaintaining glucose, cholesterol, and fatty acid metabolism (Janowski etal., 1996; Calkin and Tontonoz, 2012). Given the intricate link betweenlipid metabolism and cancer cell growth (Cairns et al., 2011), theubiquitous expression of LXRβ in melanoma is unlikely to becoincidental, allowing melanoma cells to synthesize lipids andlipoprotein particles to sustain their growth. At the same time,however, such stable basal expression levels make LXRβ an idealtherapeutic target, as exemplified by the broad-ranging responsivenessof melanoma cells to LXRβ activation therapy.

Compounds have been shown to have selectivity for LXRβ or LXRα. Thisselectivity may allow for increased activity and/or decreased off targeteffects. Examples of compounds with selectivity towards LXRβ or LXRα areshown in Table 1.

TABLE 1 EC₅₀ values for selected compounds against LXRα and LXRβCompound EC₅₀−LXRα (nM) EC₅₀−LXRβ (nM) GW3965 2 200 40 SB742881 25 74 25T0901317 1 20 50 LXR-623 3 179 24 12 <100 11 38 101-1000 630

As used herein, reference to the activity of an LXR agonist at LXRα andLXRβ refer to the activity as measured using the ligand sensing assay(LiSA) described in Spencer et al. Journal of Medicinal Chemistry 2001,44, 886-897, incorporated herein by reference. In some embodiments, theLXR agonist has an EC50 of less than 1 μM in the ligand sensing assay(e.g., 0.5 nm to 500 nM, 10 nM to 100 nM). For example, the methods ofthe invention can be performed using an LXRβ agonist having activity forLXRβ that is at least 3-fold greater than the activity of the agonistfor LXRα, or having activity for LXRβ that is at least 10-fold greaterthan the activity of the agonist for LXRα, or having activity for LXRβthat is at least 100-fold greater than the activity of said agonist forLXRα, or having activity for LXRβ that is at least within 3-fold of theactivity of the agonist for LXRα. The term “greater activity” in theLiSA assay refers to a lower EC50. For example, GW3965 2 hasapproximately 6-fold greater activity for LXRβ (EC50=30) compared toLXRα (EC50=190).

As used herein, the term “increases the level of ApoE expression invitro” refers to certain LXR agonists capable of increasing the level ofApoE expression 2.5-fold in the qPCR assay of Example 21 at aconcentration of less than 5 μM (e.g., at a concentration of 100 nM to 2μM, at a concentration of less than or equal to 1 μM). The LXR agonistsexhibiting this in vitro effect can be highly efficacious for use in themethods of the invention.

The term “alkyl” used is the present application relates a saturatedbranched or unbranched aliphatic univalent substituent. The alkylsubstituent has 1 to 100 carbon atoms, (e.g., 1 to 22 carbon atoms, 1 to10 carbon atoms 1 to 6 carbon atoms, 1 to 3 carbon atoms). Accordingly,examples of the alkyl substituent include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl andn-hexyl.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is an optionally substituted C₁-C₆ alkyl group, unless otherwisespecified. In some embodiments, the alkyl group can be substituted,e.g., the alkoxy group can have 1, 2, 3, 4, 5 or 6 substituent groups asdefined herein.

The term “alkoxyalkyl” represents a heteroalkyl group, as definedherein, that is described as an alkyl group that is substituted with analkoxy group. Exemplary unsubstituted alkoxyalkyl groups include between2 to 12 carbons. In some embodiments, the alkyl and the alkoxy each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective group.

As used herein, the term “cycloalkyl” refers to a monocyclic, bicyclic,or tricyclic substituent, which may be saturated or partially saturated,i.e. possesses one or more double bonds. Monocyclic substituents areexemplified by a saturated cyclic hydrocarbon group containing from 3 to8 carbon atoms. Examples of monocyclic cycloalkyl substituents includecyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cycloheptyl and cyclooctyl. Bicyclic fused cycloalkylsubstituents are exemplified by a cycloalkyl ring fused to anothercycloalkyl ring. Examples of bicyclic cycloalkyl substituents include,but are not limited to decalin, 1,2,3,7,8,8a-hexahydro-naphthalene, andthe like. Tricyclic cycloalkyl substituents are exemplified by acycloalkyl bicyclic fused ring fused to an additional cycloalkylsubstituent.

The term “alkylene” used is the present application relates a saturatedbranched or unbranched aliphatic bivalent substituent (e.g. the alkylenesubstituent has 1 to 6 carbon atoms, 1 to 3 carbon atoms). Accordingly,examples of the alkylene substituent include methylene, ethylene,trimethylene, propylene, tetramethylene, isopropylidene, pentamethyleneand hexamethylene.

The term “alkenylene or alkenyl” as used in the present application isan unsaturated branched or unbranched aliphatic bivalent substituenthaving a double bond between two adjacent carbon atoms (e.g. thealkenylene substituent has 2 to 6 carbon atoms, 2 to 4 carbon atoms).Accordingly, examples of the alkenylene substituent include but are notlimited to vinylene, 1-propenylene, 2-propenylene, methylvinylene,1-butenylene, 2-butenylene, 3-butenylene, 2-methyl-1-propenylene,2-methyl-2-propenylene, 2-pentenylene, 2-hexenylene.

The term “alkynylene or alkynyl” as used is the present application isan unsaturated branched or unbranched aliphatic bivalent substituenthaving a triple bond between two adjacent carbon atoms (e.g. thealkynylene substituent has 2 to 6 carbon atoms 2 to 4 carbon atoms).Examples of the alkynylene substituent include but are not limited toethynylene, 1-propynylene, 1-butynylene, 2-butynylene, 1-pentynylene,2-pentynylene, 3-pentynylene and 2-hexynylene.

The term “alkadienylene” as used is the present application is anunsaturated branched or unbranched aliphatic bivalent substituent havingtwo double bonds between two adjacent carbon atoms (e.g. thealkadienylene substituent has 4 to 10 carbon atoms). Accordingly,examples of the alkadienylene substituent include but are not limited to2,4-pentadienylene, 2,4-hexadienylene, 4-methyl-2,4-pentadienyl ene,2,4-heptadienylene, 2, 6-heptadienylene, 3-methyl-2,4-hexadienylene,2,6-octadienylene, 3-methyl-2,6-heptadienylene,2-methyl-2,4-heptadienylene, 2,8-nonadienylene,3-methyl-2,6-octadienylene, 2,6-decadienylene, 2,9-decadienylene and3,7-dimethyl-2,6-octadienylene substituents.

The term “heteroaliphatic substituent or heteroalkyl”, as used herein,refers to a monovalent or a bivalent substituent, in which one or morecarbon atoms have been substituted with a heteroatom, for instance, withan oxygen, sulfur, nitrogen, phosphorus or silicon atom, wherein thenitrogen and sulfur atoms may optionally be oxidized, and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N and Smay be placed at any interior position of the heteroaliphaticsubstituent. Examples include —CH2-CH2-O—CH3, —CH2-CH2-NH—CH3,—CH2-CH2-N(CH3)-CH3, —CH2-S—CH2-CH3, —S(O)—CH3, —CH2-CH2-S(O)2-CH3,—CH═CH—O—CH3, —CH2-CH═N—OCH3, and —CH═CH—N(CH3)-CH3. A heteroaliphaticsubstituent may be linear or branched, and saturated or unsaturated.

In one embodiment, the heteroaliphatic substituent has 1 to 100, (e.g 1to 42 carbon atoms). In yet another embodiment, the heteroaliphaticsubstituent is a polyethylene glycol residue.

As used herein, “aromatic substituent or aryl” is intended to mean anystable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atomsin each ring, wherein at least one ring is aromatic, and may beunsubstituted or substituted. Examples of such aromatic substituentsinclude phenyl, p-toluenyl (4-methylphenyl), naphthyl,tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl oracenaphthyl. In cases where the aromatic substituent is bicyclic and onering is non-aromatic, it is understood that attachment is via thearomatic ring.

The term “alkylaryl substituents or arylalkyl” refers to alkylsubstituents as described above wherein one or more bonds to hydrogencontained therein are replaced by a bond to an aryl substituent asdescribed above. It is understood that an arylalkyl substituents isconnected to the carbonyl group if the compound of the invention througha bond from the alkyl substituent. Examples of arylalkyl substituentsinclude, but are not limited to, benzyl (phenylmethyl),p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenyl ethyl,2-phenyl ethyl, 3-phenylpropyl, 2-phenylpropyl and the like.

The term “heteroaromatic substituent or heteroaryl” as used herein,represents a stable monocyclic, bicyclic or polycyclic ring of up to 10atoms in each ring, wherein at least one ring is aromatic and containsfrom 1 to 4 heteroatoms selected from the group consisting of 0, N andS. Bicyclic heteroaromatic substituents include phenyl, pyridine,pyrimidine or pyridizine rings that are

-   -   a) fused to a 6-membered aromatic (unsaturated) heterocyclic        ring having one nitrogen atom;    -   b) fused to a 5- or 6-membered aromatic (unsaturated)        heterocyclic ring having two nitrogen atoms;    -   c) fused to a 5-membered aromatic (unsaturated) heterocyclic        ring having one nitrogen atom together with either one oxygen or        one sulfur atom; or    -   d) fused to a 5-membered aromatic (unsaturated) heterocyclic        ring having one heteroatom selected from O, N or S.

Heteroaryl groups within the scope of this definition include but arenot limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl,indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl,oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl,1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl,dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The aliphatic, heteroaliphatic, aromatic and heteroaromatic substituentscan be optionally substituted one or more times, the same way ordifferently with any one or more of the following substituentsincluding, but not limited to: aliphatic, heteroaliphatic, aromatic andheteroaromatic substituents, aryl, heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; CI; Br; I; —OH; —NO2; —CN; —CF3;—CH2CF3; —CHC12; —CH2OH; —CH2CH2OH; —CH2NH2; —CH2SO2CH3; —C(O)Rx;—CO2(Rx); —CON(Rx)2; —OC(O)Rx; —OCO2Rx; —OCON(Rx)2; —N(RX)2; —S(O)Rx;—S(O)2Rx; —NRx(CO)Rx wherein each occurrence of Rx independentlyincludes, but is not limited to, aliphatic, alicyclic, heteroaliphatic,heterocyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl or heteroalkylheteroaryl, wherein anyof the aliphatic, alicyclic, heteroaliphatic, heterocyclic, alkylaryl,or alkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, saturated orunsaturated, and wherein any of the aromatic, heteroaromatic, aryl,heteroaryl, (alkyl)aryl or (alkyl)heteroaryl substituents describedabove and herein may be substituted or unsubstituted. Additionally, itwill be appreciated, that any two adjacent substituents taken togethermay represent a 4, 5, 6, or 7-membered substituted or unsubstitutedalicyclic or heterocyclic substituents. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsshown below.

The terms “halo” and “halogen” refer to a halogen atom selected from thegroup consisting of F, Cl, Br and I.

The term “halogenated alkyl substituent, haloalkyl” refers to an alkylsubstituents as defined above which is substituted with at least onehalogen atom. In an embodiment, the halogenated alkyl substituent isperhalogenated. In another embodiment, perfluoroalkyl refers to thehalogenated alkyl substituent is a univalent perfluorated substituent offormula CnF2n+1. For example, the halogenated alkyl substituent may have1 to 6 carbon atoms, (e.g. 1 to 3 carbon atoms). Accordingly, examplesof the alkyl group include trifluoromethyl, 2,2,2-trifluoroethyl,n-perfluoropropyl, n-perfluorobutyl and n-perfluoropentyl.

The term “amino,” as used herein, represents —N(RN1)2, wherein each RN1is, independently, H, OH, NO2, N(RN2)2, SO2ORN2, SO2RN2, SORN2, anN-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl,cycloalkyl, alkcycloalkyl, heterocyclyl (e.g., heteroaryl),alkheterocyclyl (e.g., alkheteroaryl), or two RN1 combine to form aheterocyclyl or an N-protecting group, and wherein each RN2 is,independently, H, alkyl, or aryl. In a preferred embodiment, amino is—NH2, or —NHRN1, wherein RN1 is, independently, OH, NO2, NH2, NRN22,SO2ORN2, SO2RN2, SORN2, alkyl, or aryl, and each RN2 can be H, alkyl, oraryl. The term “aminoalkyl,” as used herein, represents a heteroalkylgroup, as defined herein, that is described as an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group. Forexample, the alkyl moiety may comprise an oxo (═O) substituent.

As used herein, the term “aryloxy” refers to aromatic or heteroaromaticsystems which are coupled to another residue through an oxygen atom. Atypical example of an O-aryl is phenoxy. Similarly, “arylalkyl” refersto aromatic and heteroaromatic systems which are coupled to anotherresidue through a carbon chain, saturated or unsaturated, typically ofC1-C8, C₁-C₆, or more particularly C1-C4 or C1-C3 when saturated orC2-C8, C2-C6, C2-C4, or C2-C3 when unsaturated, including theheteroforms thereof. For greater certainty, arylalkyl thus includes anaryl or heteroaryl group as defined above connected to an alkyl,heteroalkyl, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moietyalso as defined above. Typical arylalkyls would be anaryl(C6-C12)alkyl(C1-C8), aryl(C6-C12)alkenyl(C2-C8), oraryl(C6-C12)alkynyl(C2-C8), plus the heteroforms. A typical example isphenylmethyl, commonly referred to as benzyl.

Typical optional substituents on aromatic or heteroaromatic groupsinclude independently halo, CN, NO2, CF3, OCF3, COOR′, CONR′2, OR′, SR′,SOR′, SO2R′, NR′2, NR′(CO)R′,NR′C(O)OR′, NR′C(O)NR′2, NR′SO2NR′2, orNR′SO2R′, wherein each R′ is independently H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as definedabove); or the substituent may be an optionally substituted groupselected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, andalkynyl groups), are typically selected from the same list ofsubstituents suitable for aromatic or heteroaromatic groups, except asnoted otherwise herein. A non-aromatic group may also include asubstituent selected from ═O and ═NOR′ where R′ is H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as definedabove).

In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl(including all heteroforms defined above) may itself optionally besubstituted by additional substituents. The nature of these substituentsis similar to those recited with regard to the substituents on the basicstructures above. Thus, where an embodiment of a substituent is alkyl,this alkyl may optionally be substituted by the remaining substituentslisted as substituents where this makes chemical sense, and where thisdoes not undermine the size limit of alkyl per se; e.g., alkylsubstituted by alkyl or by alkenyl would simply extend the upper limitof carbon atoms for these embodiments, and is not included. However,alkyl substituted by aryl, amino, halo and the like would be included.For example, where a group is substituted, the group may be substitutedwith 1, 2, 3, 4, 5, or 6 substituents. Optional substituents include,but are not limited to: C1-C6 alkyl or heteroaryl, C2-C6 alkenyl orheteroalkenyl, C2-C6 alkynyl or heteroalkynyl, halogen; aryl,heteroaryl, azido(—N3), nitro (—NO2), cyano (—CN), acyloxy(—OC(═O)R′),acyl (—C(═O)R′), alkoxy (—OR′), amido (—NR′C(═O)R″ or —C(═O)NRR′), amino(—NRR′), carboxylic acid (—CO2H), carboxylic ester (—CO2R′), carbamoyl(—OC(═O)NR′R″ or —NRC(═O)OR′), hydroxy (—OH), isocyano (—NC), sulfonate(—S(═O)20R), sulfonamide (—S(═O)2NRR′ or —NRS(═O)2R′), or sulfonyl(—S(═O)2R), where each R or R′ is selected, independently, from H, C1-C6alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl orheteroalkynyl, aryl, or heteroaryl. A substituted group may have, forexample, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituents.

The term “heterocyclyl, heterocyclic, or Het” as used herein representscyclic heteroalkyl or heteroalkenyl that is, e.g., a 3-, 4-, 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.The term “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like.

Some of the compounds of the present invention can comprise one or morestereogenic centers, and thus can exist in various isomeric forms, e.g.stereoisomers and/or diastereomers. Thus, the compounds of the inventionand pharmaceutical compositions thereof may be in the form of anindividual enantiomer, diastereomer or geometric isomer, or may be inthe form of a mixture of stereoisomers. In certain embodiments, thecompounds of the invention are enantiopure compounds. In certain otherembodiments, mixtures of stereoisomers or diastereomers are provided.Moreover, when compounds of the invention exist in tautomeric forms,each tautomer is embraced herein.

Furthermore, certain compounds, as described herein may have one or moredouble bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The invention additionally encompasses thecompounds as individual isomers substantially free of other isomers andalternatively, as mixtures of various isomers, e.g., racemic mixtures ofstereoisomers. In addition to the above-mentioned compounds per se, thisinvention also encompasses pharmaceutically acceptable derivatives ofthese compounds and compositions comprising one or more compounds of theinvention and one or more pharmaceutically acceptable excipients oradditives.

Treatment Methods

As disclosed herein, miR-1908, miR-199a-3p, miR-199a-5p, and CTGF wereidentified as endogenous metastasis promoters of metastatic invasion,endothelial recruitment, and colonization in melanoma while DNAJA4,ApoE, LRP1, LRP8, LXR, and miR7 function as metastasis suppressors orinhibitors of the same process. In addition, it was found that thesemiRNAs convergently target ApoE and the heat-shock factor DNAJA4.Cancer-secreted ApoE suppresses invasion and endothelial recruitment byactivating melanoma cell LRP1 and endothelial LRP8 receptors,respectively. DNAJA4, in turn, induces ApoE expression. These miRNAsstrongly predict human metastatic outcomes. Pre-treatment with lockednucleic acids (LNAs) targeting miR-199a-3p, miR-199a-5p, and miR-1908inhibits metastasis to multiple organs, while therapeutic delivery ofthese LNAs significantly suppresses human melanoma cell metastasis in amouse model.

Accordingly, this invention provides methods for treating melanoma viaincreasing in the subject the expression level or activity level of oneof the metastasis suppressors. This increasing can be achieved by, amongothers, forced expression of one or more of the metastasis suppressorsDNAJA4, ApoE, LRP1, and LRP8, or decreasing the expression level oractivity level of one or more miR-199a-3p, miR-199a-5p, and miR-1908. Inaddition, the treatment can be achieved by decreasing the expressionlevel or activity level of one or more of the metastasis promoters.

The invention also provides methods for treating in a subject anangiogenic disorder or a disorder of angiogenesis. The terms “angiogenicdisorder,” “disorder of angiogenesis,” and “angiogenesis disorder” areused interchangeably herein, and refer to a disorder characterized bypathological angiogenesis. A disorder characterized by pathologicalangiogenesis refers to a disorder where abnormal or aberrantangiogenesis, alone or in combination with others, contributes tocausation, origination, or symptom of the disorder. Examples of thisdisorder include various cancers (e.g., vascularized tumors), eyedisorders, inflammatory disorders, and others.

Typical vascularized tumors that can be treated with the method includesolid tumors, particularly carcinomas, which require a vascularcomponent for the provision of oxygen and nutrients. Exemplary solidtumors include, but are not limited to, carcinomas of the lung, breast,bone, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,glioblastomas, neuroblastomas, Kaposi's sarcoma, and sarcomas.

A number of disorders or conditions, other than cancer, also can betreated with the above-described method. Examples include arthritis,rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy,age-related macular degeneration, Grave's disease, vascular restenosis(including restenosis following angioplasty), arteriovenousmalformations (AVM), meningioma, hemangioma, neovascular glaucoma,chronic kidney disease, diabetic nephropathy, polycystic kidney disease,interstitial lung disease, pulmonary hypertension, chronic obstructivepulmonary disease (COPD), emphysema, autoimmune hepatitis, chronicinflammatory liver disease, hepatic cirrhosis, cutaneous T-celllymphoma, rosacea, and basal cell carcinoma.

Other treatment targets include those described in, e.g., USApplications 2009004297, 20090175791, and 20070161553, such asangiofibroma, atherosclerotic plaques, corneal graft neovascularization,hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenicgranuloma retrolental fibroplasia, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis, various other inflammatory diseasesand disorders, and endometriosis.

Forced Expression of Metastasis Suppressors

Both polypeptides of the aforementioned metastasis suppressors (e.g.,DNAJA4, ApoE, LRP1, LRP8, and LXR) and nucleic acid encoding thepolypeptides can be used to practice the invention. While manypolypeptide preparations can be used, a highly purified or isolatedpolypeptide is preferred. The terms “peptide,” “polypeptide,” and“protein” are used herein interchangeably to describe the arrangement ofamino acid residues in a polymer. A peptide, polypeptide, or protein canbe composed of the standard 20 naturally occurring amino acid, inaddition to rare amino acids and synthetic amino acid analogs. They canbe any chain of amino acids, regardless of length or post-translationalmodification (e.g., glycosylation or phosphorylation).

The polypeptide “of this invention” includes recombinantly orsynthetically produced fusion or chimeric versions of any of theaforementioned metastasis suppressors, having the particular domains orportions that are involved in the network. The term also encompassespolypeptides that have an added amino-terminal methionine (useful forexpression in prokaryotic cells).

Within the scope of this invention are fusion proteins containing one ormore of the afore-mentioned sequences and a heterologous sequence. A“chimeric” or “fusion” refers to the combination of amino acid sequencesof different origin in one polypeptide chain by in-frame combination oftheir coding nucleotide sequences. The term explicitly encompassesinternal fusions, i.e., insertion of sequences of different originwithin a polypeptide chain, in addition to fusion to one of its termini.A heterologous polypeptide, nucleic acid, or gene is one that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its original form. Two fused domains or sequences areheterologous to each other if they are not adjacent to each other in anaturally occurring protein or nucleic acid.

An “isolated” or “purified” polypeptide refers to a polypeptide that hasbeen separated from other proteins, lipids, and nucleic acids with whichit is naturally associated. The polypeptide can constitute at least 10%(i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purifiedpreparation. Purity can be measured by any appropriate standard method,for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. An isolated polypeptide described inthe invention can be purified from a natural source, produced byrecombinant DNA techniques, or by chemical methods.

A “recombinant” polypeptide refers to a polypeptide produced byrecombinant DNA techniques; i.e., produced from cells transformed by anexogenous DNA construct encoding the desired polypeptide. A “synthetic”polypeptide refers to a polypeptide prepared by chemical synthesis. Theterm “recombinant” when used with reference, e.g., to a cell, nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified.

“Overexpression” refers to the expression of a RNA or polypeptideencoded by a nucleic acid introduced into a host cell, wherein the RNAor polypeptide or protein is either not normally present in the hostcell, or wherein the RNA or polypeptide is present in said host cell ata higher level than that normally expressed from the endogenous geneencoding the RNA or polypeptide.

The amino acid composition of each of the above-mentioned polypeptidesmay vary without disrupting their functions—the ability to up-regulatethe above-mentioned network (e.g., increase the activation level of theApoE/LRP signaling pathway), thereby inhibiting metastasis to multipleorgans. For example, it can contain one or more conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan), β-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in one of the above-describedpolypeptides (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18) ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, mutations can be introduced randomly alongall or part of the sequences, such as by saturation mutagenesis, and theresultant mutants can be screened for the ability to up-regulate theabove-mentioned network or ApoE/LRP signaling pathway, and trigger therespective cellular response to identify mutants that retain theactivity as descried below in the examples.

A functional equivalent of a polypeptide of this invention refers to aderivative of the polypeptide, e.g., a protein having one or more pointmutations, insertions, deletions, truncations, a fusion protein, or acombination thereof. It retains substantially the activity to of theabove-mentioned polypeptide. The isolated polypeptide of this inventioncan contain the sequence of one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, and 18, or a functional equivalent or fragment thereof. In general,the functional equivalent is at least 75% (e.g., any number between 75%and 100%, inclusive, e.g., 70%, 80%, 85%, 90%, 95%, and 99%) identicalto one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

A polypeptide described in this invention can be obtained as arecombinant polypeptide. To prepare a recombinant polypeptide, a nucleicacid encoding it can be linked to another nucleic acid encoding a fusionpartner, e.g., glutathione-s-transferase (GST), 6×-His epitope tag, orM13 Gene 3 protein. The resultant fusion nucleic acid expresses insuitable host cells a fusion protein that can be isolated by methodsknown in the art. The isolated fusion protein can be further treated,e.g., by enzymatic digestion, to remove the fusion partner and obtainthe recombinant polypeptide of this invention. Alternatively, thepolypeptide of the invention can be chemically synthesized (see e.g.,Creighton, “Proteins: Structures and Molecular Principles,” W.H. Freeman& Co., NY, 1983). For additional guidance, skilled artisans may consultAusubel et al. (Current Protocols in Molecular Biology and ShortProtocols in Molecular Biology, 3rd Ed. 1987 & 1995), Sambrook et al.(Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989), and chemical synthesis Gait, M. J. Ed.(Oligonucleotide Synthesis, IRL Press, Oxford, 1984).

Due to their functions as cellular protein or membrane protein, DNAJA4,LRP1, LRP8, and LXR can be associated with, e.g., conjugated or fusedto, one or more of an amino acid sequence comprising a cell-penetratingpeptide (CPP) sequence, and the like. In this manner, a composition ofthe invention as discussed below can include a transport enhancer. Acell-penetrating peptide (CPP) generally consists of less than 30 aminoacids and has a net positive charge. CPPs internalize in living animalcells in an endocytotic or receptor/energy-independent manner. There areseveral classes of CPPs with various origins, from totallyprotein-derived CPPs via chimeric CPPs to completely synthetic CPPs.Examples of CPPs are known in the art. See, e.g., U.S. Application Nos.20090099066 and 20100279918. It is know that CPPs can delivery anexogenous protein into various cells.

All of naturally occurring versions, genetic engineered versions, andchemically synthesized versions of the above-mentioned polypeptides canbe used to practice the invention disclosed therein. Polypeptidesobtained by recombinant DNA technology may have the same amino acidsequence as a naturally occurring version (e.g., one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, and 18) or a functionally equivalent thereof.They also include chemically modified versions. Examples of chemicallymodified polypeptides include polypeptides subjected to conformationalchange, addition or deletion of a side chain, and those to which acompound such as polyethylene glycol has been bound. Once purified andtested by standard methods or according to the method described in theexamples below or other methods known in the art, the polypeptides canbe included in suitable composition.

For expressing the above-mentioned factors, the invention provides anucleic acid that encodes any of the polypeptides mentioned above.Preferably, the nucleotide sequences are isolated and/or purified. Anucleic acid refers to a DNA molecule (e.g., but not limited to, a cDNAor genomic DNA), an RNA molecule (e.g., but not limited to, an mRNA), ora DNA or RNA analog. A DNA or RNA analog can be synthesized fromnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded. An “isolated nucleic acid” is a nucleic acid thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid. The term therefore covers, for example, (a) a DNA whichhas the sequence of part of a naturally occurring genomic DNA moleculebut is not flanked by both of the coding sequences that flank that partof the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein.

The terms “RNA,” “RNA molecule,” and “ribonucleic acid molecule” areused interchangeably herein, and refer to a polymer of ribonucleotides.The term “DNA” or “DNA molecule” or deoxyribonucleic acid molecule”refers to a polymer of deoxyribonucleotides. DNA and RNA can besynthesized naturally (e.g., by DNA replication or transcription of DNA,respectively). RNA can be post-transcriptionally modified. DNA and RNAalso can be chemically synthesized. DNA and RNA can be single-stranded(i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g.,double-stranded, i.e., dsRNA and dsDNA, respectively).

The present invention also provides recombinant constructs having one ormore of the nucleotide sequences described herein. Example of theconstructs include a vector, such as a plasmid or viral vector, intowhich a nucleic acid sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred embodiment, the constructfurther includes regulatory sequences, including a promoter, operablylinked to the sequence. Large numbers of suitable vectors and promotersare known to those of skill in the art, and are commercially available.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are also described in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press).

Examples of expression vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of or Simian virus 40 (SV40),bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, a nucleic acid sequenceencoding one of the polypeptides described above can be inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and related sub-cloning procedures are within thescope of those skilled in the art.

The nucleic acid sequence in the aforementioned expression vector ispreferably operatively linked to an appropriate transcription controlsequence (promoter) to direct mRNA synthesis. Examples of such promotersinclude: the retroviral long terminal (LTR) or SV40 promoter, the E.coli lac or trp promoter, the phage lambda PL promoter, and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or viruses. The expression vector can also contain aribosome binding site for translation initiation, and a transcriptionterminator. The vector may include appropriate sequences for amplifyingexpression. In addition, the expression vector preferably contains oneor more selectable marker genes to provide a phenotypic trait forselection of transformed host cells such as dihydrofolate reductase orneomycin resistance for eukaryotic cell cultures, or such astetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate nucleic acid sequences asdescribed above, as well as an appropriate promoter or control sequence,can be employed to transform an appropriate host to permit the host toexpress the polypeptides described above. Such vectors can be used ingene therapy. Examples of suitable expression hosts include bacterialcells (e.g., E. coli, Streptomyces, Salmonella typhimurium), fungalcells (yeast), insect cells (e.g., Drosophila and Spodoptera frugiperda(519)), animal cells (e.g., CHO, COS, and HEK 293), adenoviruses, andplant cells. The selection of an appropriate host is within the scope ofthose skilled in the art. In some embodiments, the present inventionprovides methods for producing the above mentioned polypeptides bytransfecting a host cell with an expression vector having a nucleotidesequence that encodes one of the polypeptides. The host cells are thencultured under a suitable condition, which allows for the expression ofthe polypeptide.

Decreasing Expression or Activity Level of Metastasis Promoters

As mentioned above, one can use an inhibitory agent that decreases theexpression or activity level of miR-199a-3p, miR-199a-5p, miR-1908, orCTGF in treating melanoma. An inhibitory agent (i.e., inhibitor) can bea nucleic acid, a polypeptide, an antibody, or a small moleculecompound. In one example, the inhibitor functions at a level oftranscription, mRNA stability, translation, proteinstability/degradation, protein modification, and protein binding.

A nucleic acid inhibitor can encode a small interference RNA (e.g., anRNAi agent) that targets one or more of the above-mentioned genes, e.g.,CTGF, and inhibits its expression or activity. The term “RNAi agent”refers to an RNA, or analog thereof, having sufficient sequencecomplementarity to a target RNA to direct RNA interference. Examplesalso include a DNA that can be used to make the RNA. RNA interference(RNAi) refers to a sequence-specific or selective process by which atarget molecule (e.g., a target gene, protein or RNA) is down-regulated.Generally, an interfering RNA (“iRNA”) is a double strandedshort-interfering RNA (siRNA), short hairpin RNA (shRNA), orsingle-stranded micro-RNA (miRNA) that results in catalytic degradationof specific mRNAs, and also can be used to lower or inhibit geneexpression.

The term “short interfering RNA” or “siRNA” (also known as “smallinterfering RNAs”) refers to an RNA agent, preferably a double-strandedagent, of about 10-50 nucleotides in length, preferably between about15-25 nucleotides in length, more preferably about 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length, the strands optionally havingoverhanging ends comprising, for example 1, 2 or 3 overhangingnucleotides (or nucleotide analogs), which is capable of directing ormediating RNA interference. Naturally-occurring siRNAs are generatedfrom longer dsRNA molecules (e.g., >25 nucleotides in length) by acell's RNAi machinery (e.g., Dicer or a homolog thereof).

The term “miRNA” or “microRNA” refers to an RNA agent, preferably asingle-stranded agent, of about 10-50 nucleotides in length, preferablybetween about 15-25 nucleotides in length, more preferably about 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length, which is capable ofdirecting or mediating RNA interference. Naturally-occurring miRNAs aregenerated from stem-loop precursor RNAs (i.e., pre-miRNAs) by Dicer. Theterm “Dicer” as used herein, includes Dicer as well as any Dicerorthologue or homologue capable of processing dsRNA structures intosiRNAs, miRNAs, siRNA-like or miRNA-like molecules. The term microRNA(or “miRNA”) is used interchangeably with the term “small temporal RNA”(or “stRNA”) based on the fact that naturally-occurring microRNAs (or“miRNAs”) have been found to be expressed in a temporal fashion (e.g.,during development).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region.

Within the scope of this invention is utilization of RNAi featuringdegradation of RNA molecules (e.g., within a cell). Degradation iscatalyzed by an enzymatic, RNA-induced silencing complex (RISC). A RNAagent having a sequence sufficiently complementary to a target RNAsequence (e.g., the above-mentioned CTGF gene) to direct RNAi means thatthe RNA agent has a homology of at least 50%, (e.g., 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% homology) to the target RNA sequence so thatthe two are sufficiently complementary to each other to hybridize andtrigger the destruction of the target RNA by the RNAi machinery (e.g.,the RISC complex) or process. A RNA agent having a “sequencesufficiently complementary to a target RNA sequence to direct RNAi” alsomeans that the RNA agent has a sequence sufficient to trigger thetranslational inhibition of the target RNA by the RNAi machinery orprocess. A RNA agent also can have a sequence sufficiently complementaryto a target RNA encoded by the target DNA sequence such that the targetDNA sequence is chromatically silenced. In other words, the RNA agenthas a sequence sufficient to induce transcriptional gene silencing,e.g., to down-modulate gene expression at or near the target DNAsequence, e.g., by inducing chromatin structural changes at or near thetarget DNA sequence.

The above-mentioned polynucleotides can be delivered using polymeric,biodegradable microparticle or microcapsule delivery devices known inthe art. Another way to achieve uptake of the polynucleotides is usingliposomes, prepared by standard methods. The polynucleotide can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano, etal., 1995, J. Mol. Med. 73:479). Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements that are known in the art. Delivery of naked DNA(i.e., without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site is another means to achieve in vivo expression.

siRNA, miRNA, and asRNA (antisense RNA) molecules can be designed bymethods well known in the art. siRNA, miRNA, and asRNA molecules withhomology sufficient to provide sequence specificity required to uniquelydegrade any RNA can be designed using programs known in the art,including, but not limited to, those maintained on websites for AMBION,Inc. and DHARMACON, Inc. Systematic testing of several designed speciesfor optimization of the siRNA, miRNA, and asRNA sequence can beroutinely performed by those skilled in the art. Considerations whendesigning short interfering nucleic acid molecules include, but are notlimited to, biophysical, thermodynamic, and structural considerations,base preferences at specific positions in the sense strand, andhomology. These considerations are well known in the art and provideguidelines for designing the above-mentioned RNA molecules.

An antisense polynucleotide (preferably DNA) of the present inventioncan be any antisense polynucleotide so long as it possesses a basesequence complementary or substantially complementary to that of thegene encoding a component of the aforementioned network. The basesequence can be at least about 70%, 80%, 90%, or 95% homology to thecomplement of the gene encoding the polypeptide. These antisense DNAscan be synthesized using a DNA synthesizer.

The antisense DNA of the present invention may contain changed ormodified sugars, bases or linkages. The antisense DNA, as well as theRNAi agent mentioned above, may also be provided in a specialized formsuch as liposomes, microspheres, or may be applied to gene therapy, ormay be provided in combination with attached moieties. Such attachedmoieties include polycations such as polylysine that act as chargeneutralizers of the phosphate backbone, or hydrophobic moieties such aslipids (e.g., phospholipids, cholesterols, etc.) that enhance theinteraction with cell membranes or increase uptake of the nucleic acid.Preferred examples of the lipids to be attached are cholesterols orderivatives thereof (e.g., cholesteryl chloroformate, cholic acid,etc.). These moieties may be attached to the nucleic acid at the 3′ or5′ ends thereof and may also be attached thereto through a base, sugar,or intramolecular nucleoside linkage. Other moieties may be cappinggroups specifically placed at the 3′ or 5′ ends of the nucleic acid toprevent degradation by nucleases such as exonuclease, RNase, etc. Suchcapping groups include, but are not limited to, hydroxyl protectinggroups known in the art, including glycols such as polyethylene glycol,tetraethylene glycol and the like. The inhibitory action of theantisense DNA can be examined using a cell-line or animal based geneexpression system of the present invention in vivo and in vitro.

The above-discussed nucleic acids encoding one or more of thepolypeptides mentioned above or RNAi agents can be cloned in a vectorfor delivering to cells in vitro or in vivo. For in vivo uses, thedelivery can target a specific tissue or organ (e.g., skin). Targeteddelivery involves the use of vectors (e.g., organ-homing peptides) thatare targeted to specific organs or tissues after systemicadministration. For example, the vector can have a covalent conjugate ofavidin and a monoclonal antibody to a liver specific protein.

In certain embodiments, the present invention provides methods for invivo expression the above-mentioned metastsis suppressors. Such methodwould achieve its therapeutic effect by introduction of nucleic acidsequences encoding any of the factors into cells or tissues of a humanor a non-human animal in need of inhibiting endothelial recruitment,cancer cell invasion, or metastatic angiogenesis. Delivery of thenucleic acid sequences can be achieved using a recombinant expressionvector such as a chimeric virus or a colloidal dispersion system.Preferred for therapeutic delivery of the nucleic acid sequences is theuse of targeted liposomes.

Various viral vectors which can be utilized for gene therapy disclosedherein include, adenovirus, adeno-associated virus (AAV), herpes virus,vaccinia, or, preferably, an RNA virus such as a retrovirus and alentivirus. Preferably, the retroviral vector is a lentivirus or aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes.

All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using a target-specific antibody or hormone that has areceptor in the target. Those of skill in the art will recognize thatspecific polynucleotide sequences can be inserted into the retroviralgenome or attached to a viral envelope to allow target specific deliveryof the retroviral vector.

Another targeted system for delivery of nucleic acids is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. RNA, DNA, and intact virions canbe encapsulated within the aqueous interior and delivered to cells in abiologically active form. Methods for efficient gene transfer using aliposome vehicle are known in the art. The composition of the liposomeis usually a combination of phospholipids, usually in combination withsteroids, especially cholesterol. Other phospholipids or other lipidsmay also be used. The physical characteristics of liposomes depend onpH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidyl-ethanolamine, sphingolipids,cerebrosides, and gangliosides. Exemplary phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoyl-phosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

When used in vivo, it is desirable to use a reversibledelivery-expression system. To that end, the Cre-loxP or FLP/FRT systemand other similar systems can be used for reversible delivery-expressionof one or more of the above-described nucleic acids. See WO2005/112620,WO2005/039643, U.S. Applications 20050130919, 20030022375, 20020022018,20030027335, and 20040216178. In particular, the reversibledelivery-expression system described in US Application NO 20100284990can be used to provide a selective or emergency shut-off.

In another example, the above-mentioned inhibitory agent can be apolypeptide or a protein complex, such as an antibody. The term“antibody” refers to an immunoglobulin molecule or immunologicallyactive portion thereof, i.e., an antigen-binding portion. Examplesinclude, but are not limited to, a protein having at least one or two,heavy (H) chain variable regions (VH), and at least one or two light (L)chain variable regions (VL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). As used herein, the term“immunoglobulin” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized human immunoglobulin genes include the kappa, lambda, alpha(IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon andmu constant region genes, as well as the myriad immunoglobulin variableregion genes.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., LRP1, LRP8, and CTGF).It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

Antibodies that specifically bind to one of the above-mentioned targetproteins (e.g., CTGF) can be made using methods known in the art. Thisantibody can be a polyclonal or a monoclonal antibody. In oneembodiment, the antibody can be recombinantly produced, e.g., producedby phage display or by combinatorial methods. In another embodiment, theantibody is a fully human antibody (e.g., an antibody made in a mousewhich has been genetically engineered to produce an antibody from ahuman immunoglobulin sequence), a humanized antibody, or a non-humanantibody, for example, but not limited to, a rodent (mouse or rat),goat, primate (for example, but not limited to, monkey), rabbit, orcamel antibody. Examples of methods to generate humanized version ofantibodies include, but are not limited to, CDR grafting (Queen et al.,U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)), chainshuffling (U.S. Pat. No. 5,565,332); and veneering or resurfacing (EP592,106; EP 519,596); Padlan, Molecular Immunology 28(415):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)). Examples of methods togenerate fully human antibodies include, but are not limited to,generation of antibodies from mice that can express human immunoglobulingenes and use of phage-display technology to generate and screen humanimmunoglobulin gene libraries.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds CTGFis substantially free of antibodies that specifically bind antigensother than such an antigen). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes. Thephrases “an antibody recognizing an antigen” and “an antibody specificfor an antigen” are used interchangeably herein with the term “anantibody which binds specifically to an antigen.” As used herein, theterm “high affinity” for an IgG antibody refers to an antibody having aKD of 10-7 M or less, preferably 10-8 M or less, more preferably 10-9 Mor less and even more preferably 10-10 M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a KD of 10-7 M or less, more preferably 10-8 M or less.

In one example, a composition contains a monoclonal antibody thatneutralizes CTGF. In one embodiment, this antibody can be a fully humanantibody, a humanized antibody, or a non-human antibody, for example,but not limited to, a rodent (mouse or rat), goat, primate (for example,but not limited to, monkey), rabbit, or camel antibody. In oneembodiment, one or more amino-acids of this monoclonal monoclonalantibody may be substituted in order to alter its physical properties.These properties include, but are not limited to, binding specificity,binding affinity, immunogenicity, and antibody isotype. Pharmaceuticalcompositions containing fully human or humanized versions of the abovedescribed antibodies can be used for treating melanoma or for inhibitingendothelial recruitment, cancer cell invasion, or metastaticangiogenesis.

As used herein, a “subject” refers to a human and a non-human animal.Examples of a non-human animal include all vertebrates, e.g., mammals,such as non-human mammals, non-human primates (particularly higherprimates), dog, rodent (e.g., mouse or rat), guinea pig, cat, andrabbit, and non-mammals, such as birds, amphibians, reptiles, etc. Inone embodiment, the subject is a human. In another embodiment, thesubject is an experimental animal or animal suitable as a disease model.A subject to be treated for a disorder can be identified by standarddiagnosing techniques for the disorder. Optionally, the subject can beexamined for mutation, expression level, or activity level of one ormore of the miR-199a-3p, miR-199a-5p, miR-1908, and CTGF mentioned aboveby methods known in the art or described above before treatment. If thesubject has a particular mutation in the gene, or if the gene expressionor activity level is, for example, greater in a sample from the subjectthan that in a sample from a normal person, the subject is a candidatefor treatment of this invention.

To confirm the inhibition or treatment, one can evaluate and/or verifythe inhibition of endothelial recruitment or resulting angiogenesisusing technology known in the art before and/or after the administeringstep. Exemplary technologies include angiography or arteriography, amedical imaging technique used to visualize the inside, or lumen, ofblood vessels and organs of the body, can generally be done by injectinga radio-opaque contrast agent into the blood vessel and imaging usingX-ray based techniques such as fluoroscopy.

“Treating” or “treatment” as used herein refers to administration of acompound or agent to a subject who has a disorder with the purpose tocure, alleviate, relieve, remedy, delay the onset of, prevent, orameliorate the disorder, the symptom of a disorder, the disease statesecondary to the disorder, or the predisposition toward the disorder. An“effective amount” or “therapeutically effective amount” refers to anamount of the compound or agent that is capable of producing a medicallydesirable result in a treated subject. The treatment method can beperformed in vivo or ex vivo, alone or in conjunction with other drugsor therapy. A therapeutically effective amount can be administered inone or more administrations, applications or dosages and is not intendedto be limited to a particular formulation or administration route.

The expression “effective amount” as used herein, refers to a sufficientamount of the compound of the invention to exhibit the desiredtherapeutic effect. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the particular therapeutic agent and the like. The compounds ofthe invention are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit of therapeuticagent appropriate for the patient to be treated. It will be understood,however, that the total daily usage of the compounds and compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient or organism will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the anticancer activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

A therapeutic agent can be administered in vivo or ex vivo, alone orco-administered in conjunction with other drugs or therapy, i.e., acocktail therapy. As used herein, the term “co-administration” or“co-administered” refers to the administration of at least two agent(s)or therapies to a subject. For example, in the treatment of tumors,particularly vascularized, malignant tumors, the agents can be usedalone or in combination with, e.g., chemotherapeutic, radiotherapeutic,apoptopic, anti-angiogenic agents and/or immunotoxins or coaguligands.In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents/therapies used may vary.

In an in vivo approach, a compound or agent is administered to asubject. Generally, the compound is suspended in apharmaceutically-acceptable carrier (such as, for example, but notlimited to, physiological saline) and administered orally or byintravenous infusion, or injected or implanted subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compounds available and the different efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the compound in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) can increase theefficiency of delivery, particularly for oral delivery.

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the therapeutic agents describedabove. The composition can be a pharmaceutical composition that containsa pharmaceutically acceptable carrier, a dietary composition thatcontains a dietarily acceptable suitable carrier, or a cosmeticcomposition that contains a cosmetically acceptable carrier.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active compound.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,sodium lauryl sulfate, and D&C Yellow #10.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting a free base or free acid function with a suitable reagent, asdescribed generally below. For example, a free base function can bereacted with a suitable acid. Furthermore, where the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof may, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts, include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; natural and synthetic phospholipids, such as soybean andegg yolk phosphatides, lecithin, hydrogenated soy lecithin, dimyristoyllecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleoyl lecithin,hydroxylated lecithin, lysophosphatidylcholine, cardiolipin,sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine,diastearoyl phosphatidylethanolamine (DSPE) and its pegylated esters,such as DSPE-PEG750 and, DSPE-PEG2000, phosphatidic acid, phosphatidylglycerol and phosphatidyl serine. Commercial grades of lecithin whichare preferred include those which are available under the trade namePhosal® or Phospholipon® and include Phosal 53 MCT, Phosal 50 PG, Phosal75 SA, Phospholipon 90H, Phospholipon 90G and Phospholipon 90 NG;soy-phosphatidylcholine (SoyPC) and DSPE-PEG2000 are particularlypreferred; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The above-described composition, in any of the forms described above,can be used for treating melanoma, or any other disease or conditiondescribed herein. An effective amount refers to the amount of an activecompound/agent that is required to confer a therapeutic effect on atreated subject. Effective doses will vary, as recognized by thoseskilled in the art, depending on the types of diseases treated, route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatment.

A pharmaceutical composition of this invention can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intrmuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent. Such solutionsinclude, but are not limited to, 1,3-butanediol, mannitol, water,Ringer's solution, and isotonic sodium chloride solution. In addition,fixed oils are conventionally employed as a solvent or suspending medium(e.g., synthetic mono- or diglycerides). Fatty acid, such as, but notlimited to, oleic acid and its glyceride derivatives, are useful in thepreparation of injectables, as are natural pharmaceutically acceptableoils, such as, but not limited to, olive oil or castor oil,polyoxyethylated versions thereof. These oil solutions or suspensionsalso can contain a long chain alcohol diluent or dispersant such as, butnot limited to, carboxymethyl cellulose, or similar dispersing agents.Other commonly used surfactants, such as, but not limited to, Tweens orSpans or other similar emulsifying agents or bioavailability enhancers,which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms also can be used for thepurpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include, but are not limited to, lactose and cornstarch. Lubricating agents, such as, but not limited to, magnesiumstearate, also are typically added. For oral administration in a capsuleform, useful diluents include, but are not limited to, lactose and driedcorn starch. When aqueous suspensions or emulsions are administeredorally, the active ingredient can be suspended or dissolved in an oilyphase combined with emulsifying or suspending agents. If desired,certain sweetening, flavoring, or coloring agents can be added.

Pharmaceutical compositions for topical administration according to thedescribed invention can be formulated as solutions, ointments, creams,suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils.Alternatively, topical formulations can be in the form of patches ordressings impregnated with active ingredient(s), which can optionallycomprise one or more excipients or diluents. In some preferredembodiments, the topical formulations include a material that wouldenhance absorption or penetration of the active agent(s) through theskin or other affected areas.

A topical composition contains a safe and effective amount of adermatologically acceptable carrier suitable for application to theskin. A “cosmetically acceptable” or “dermatologically-acceptable”composition or component refers a composition or component that issuitable for use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Thecarrier enables an active agent and optional component to be deliveredto the skin at an appropriate concentration(s). The carrier thus can actas a diluent, dispersant, solvent, or the like to ensure that the activematerials are applied to and distributed evenly over the selected targetat an appropriate concentration. The carrier can be solid, semi-solid,or liquid. The carrier can be in the form of a lotion, a cream, or agel, in particular one that has a sufficient thickness or yield point toprevent the active materials from sedimenting. The carrier can be inertor possess dermatological benefits. It also should be physically andchemically compatible with the active components described herein, andshould not unduly impair stability, efficacy, or other use benefitsassociated with the composition.

Combination Therapies

In some embodiments, the pharmaceutical composition may further comprisean additional compound having antiproliferative activity. The additionalcompound having antiproliferative activity can be selected from a groupof antiproliferative agents including those shown in Table 2.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder, or they may achieve different effects(e.g., control of any adverse effects).

By “antiproliferative agent” is meant any antiproliferative agent,including those antiproliferative agents listed in Table 2, any of whichcan be used in combination with a LXR agonist to treat the medicalconditions recited herein. Antiproliferative agents also includeorgano-platine derivatives, naphtoquinone and benzoquinone derivatives,chrysophanic acid and anthroquinone derivatives thereof.

TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazineifosfamide altretamine hexamethylmelamine estramustine phosphatethiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomidecyclophosphamide Semustine Platinum agents spiroplatin lobaplatin(Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464(Hoffmann-La iproplatin Roche) ZD-0473 (AnorMED) SM-11355 (Sumitomo)oxaliplatin AP-5280 (Access) carboplatin cisplatin Antimetabolitesazacytidine trimetrexate Floxuridine deoxycoformycin2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision)2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC(Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabinegemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecanmesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposidegimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan(Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS -103 (Taiho)dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone(Novuspharma) J-107088 (Merck & Co) rebeccamycin analogue (Exelixis)BNP-1350 (BioNumerik) BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang)rubitecan (SuperGen) KW-2170 (Kyowa Hakko) irinotecan (CPT-11)hydroxycamptothecin (SN-38) topotecan Antitumor valrubicin azonafideantibiotics therarubicin anthrapyrazole idarubicin oxantrazolerubidazone losoxantrone plicamycin MEN-10755 (Menarini) porfiromycinGPX-100 (Gem mitoxantrone (novantrone) Pharmaceuticals) amonafideEpirubicin mitoxantrone doxorubicin Antimitotic colchicine E7010(Abbott) agents vinblastine PG-TXL (Cell Therapeutics) vindesine IDN5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin(BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis)isohomohalichondrin-B T 900607 (Tularik) (PharmaMar) T 138067 (Tularik)ZD 6126 (AstraZeneca) cryptophycin 52 (Eli Lilly) AZ10992 (Asahi)vinflunine (Fabre) IDN-5109 (Indena) auristatin PE (Teikoku Hormone)AVLB (Prescient NeuroPharma) BMS 247550 (BMS) azaepothilone B (BMS) BMS184476 (BMS) BNP-7787 (BioNumerik) BMS 188797 (BMS) CA-4 prodrug(OXiGENE) taxoprexin (Protarga) dolastatin-10 (NIH) SB 408075(GlaxoSmithKline) CA-4 (OXiGENE) Vinorelbine docetaxel Trichostatin Avincristine paclitaxel Aromatase aminoglutethimide YM-511 (Yamanouchi)inhibitors atamestane (BioMedicines) formestane letrozole exemestaneanastrazole Thymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias)synthase inhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagoniststrabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxtermafosfamide (Baxter International) International) albumin + 32P (Isotopeapaziquone (Spectrum Solutions) Pharmaceuticals) thymectacin(NewBiotics) O6 benzyl guanine (Paligent) Farnesyltransferase arglabin(NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib(Schering-Plough) perillyl alcohol (DOR BAY-43-9006 (Bayer) BioPharma)Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (Elitariquidar (Xenova) Lilly) MS-209 (Schering AG) biricodar dicitrate(Vertex) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrateacetyltransferase SAHA (Aton Pharma) (Titan) inhibitors MS-275 (ScheringAG) depsipeptide (Fujisawa) Metalloproteinase Neovastat (AeternaLaboratories) CMT-3 (CollaGenex) inhibitors marimastat (British Biotech)BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan)tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox(Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid(Celgene) agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan(Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca)Retinoic acid fenretinide (Johnson & Johnson) alitretinoin (Ligand)receptor agonists LGD-1550 (Ligand) Immuno- interferon dexosome therapy(Anosys) modulators oncophage (Antigenics) pentrix (Australian CancerGMK (Progenics) Technology) adenocarcinoma vaccine ISF-154 (Tragen)(Biomira) cancer vaccine (Intercell) CTP-37 (AVI BioPharma) norelin(Biostar) IRX-2 (Immuno-Rx) BLP-25 (Biomira) PEP-005 (Peplin Biotech)MGV (Progenics) synchrovax vaccines (CTL β-alethine (Dovetail) Immuno)CLL therapy (Vasogen) melanoma vaccine (CTL Ipilimumab (BMS), Immuno)CM-10 (cCam Biotherapeutics) p21 RAS vaccine (GemVax) MPDL3280A(Genentech) MAGE-A3 (GSK) nivolumab (BMS) abatacept (BMS) Hormonal andestrogens dexamethasone antihormonal conjugated estrogens prednisoneagents ethinyl estradiol methylprednisolone chlortrianisen prednisoloneidenestrol aminoglutethimide hydroxyprogesterone caproate leuprolidemedroxyprogesterone octreotide testosterone mitotane testosteronepropionate; P-04 (Novogen) fluoxymesterone 2-methoxyestradiol (EntreMed)methyltestosterone arzoxifene (Eli Lilly) diethylstilbestrol tamoxifenmegestrol toremofine bicalutamide goserelin flutamide Leuporelinnilutamide bicalutamide Photodynamic talaporfin (Light Sciences)Pd-bacteriopheophorbide (Yeda) agents Theralux (Theratechnologies)lutetium texaphyrin motexafin gadolinium (Pharmacyclics) (Pharmacyclics)hypericin Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth)leflunomide (Sugen/Pharmacia) kahalide F (PharmaMar) ZD1839(AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751(Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera)PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668(Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech)ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4(Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline)ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumab(Genentech) Tyrphostins OSI-774 (Tarceva ™) Gefitinib (Iressa) CI-1033(Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3(York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B-Rafenzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo) SR-27897 (CCK Ainhibitor, Sanofi- ceflatonin (apoptosis promotor, Synthelabo)ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BCX-1777 (PNPinhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase(ribonuclease stimulant, CV-247 (COX-2 inhibitor, Ivy Medical) Alfacell)P54 (COX-2 inhibitor, Phytopharm) galarubicin (RNA synthesis inhibitor,CapCell ™ (CYP450 stimulant, Bavarian Dong-A) Nordic) tirapazamine(reducing agent, SRI GCS-100 (gal3 antagonist, GlycoGenesys)International) G17DT immunogen (gastrin inhibitor, N-acetylcysteine(reducing agent, Aphton) Zambon) efaproxiral (oxygenator, AllosTherapeutics) R-flurbiprofen (NF-kappaB inhibitor, PI-88 (heparanaseinhibitor, Progen) Encore) tesmilifene (histamine antagonist, YM 3CPA(NF-kappaB inhibitor, Active BioSciences) Biotech) histamine (histamineH2 receptor agonist, seocalcitol (vitamin D receptor agonist, Maxim)Leo) tiazofurin (IMPDH inhibitor, Ribapharm) 131-I-TM-601 (DNAantagonist, cilengitide (integrin antagonist, Merck KGaA)TransMolecular) SR-31747 (IL-1 antagonist, Sanofi- eflornithine (ODCinhibitor, ILEX Synthelabo) Oncology) CCI-779 (mTOR kinase inhibitor,Wyeth) minodronic acid (osteoclast inhibitor, exisulind (PDE Vinhibitor, Cell Pathways) Yamanouchi) CP-461 (PDE V inhibitor, CellPathways) indisulam (p53 stimulant, Eisai) AG-2037 (GART inhibitor,Pfizer) aplidine (PPT inhibitor, PharmaMar) WX-UK1 (plasminogenactivator inhibitor, gemtuzumab (CD33 antibody, Wyeth Wilex) Ayerst)PBI-1402 (PMN stimulant, ProMetic PG2 (hematopoiesis enhancer,LifeSciences) Pharmagenesis) bortezomib (proteasome inhibitor, Immunol ™(triclosan oral rinse, Endo) Millennium) triacetyluri dine (uridineprodrug, SRL-172 (T cell stimulant, SR Pharma) Wellstat) TLK-286(glutathione S transferase inhibitor, SN-4071 (sarcoma agent, SignatureTelik) BioScience) PT-100 (growth factor agonist, Point TransMID-107 ™(immunotoxin, KS Therapeutics) Biomedix) midostaurin (PKC inhibitor,Novartis) PCK-3145 (apoptosis promotor, Procyon) bryostatin-1 (PKCstimulant, GPC Biotech) doranidazole (apoptosis promotor, Pola) CDA-II(apoptosis promotor, Everlife) CHS-828 (cytotoxic agent, Leo) SDX-101(apoptosis promotor, Salmedix) trans-retinoic acid (differentiator, NIH)rituximab (CD20 antibody, Genentech MX6 (apoptosis promotor, MAXIA)carmustine apomine (apoptosis promotor, ILEX Mitoxantrone Oncology)Bleomycin urocidin (apoptosis promotor, Bioniche) Absinthin Ro-31-7453(apoptosis promotor, La Chrysophanic acid Roche) Cesium oxidesbrostallicin (apoptosis promotor, BRAF inhibitors, Pharmacia) PDL1inhibitors β-lapachone MEK inhibitors gelonin

indicates data missing or illegible when filed

Diagnosis and Prognosis Methods

The above-describe genes can be used in determining whether a subjecthas, or is at risk of having, metastatic melanoma. Alternatively, theycan be used for determining a prognosis of such a disorder in a subject.

Diagnosis Methods

In one aspect, the invention provides qualitative and quantitativeinformation to determine whether a subject has or is predisposed tometastatic melanoma or other disease characterized by endothelialrecruitment, cancer cell invasion, or metastatic angiogenesis. A subjecthaving such a disorder or prone to it can be determined based on theexpression levels, patterns, or profiles of the above-described genes ortheir products (mRNAs, microRNAs, or polypeptides) in a test sample fromthe subject. In other words, the products can be used as markers toindicate the presence or absence of the disorder. Diagnostic andprognostic assays of the invention include methods for assessing theexpression level of the products. The methods allow one to detect thedisorder. For example, a relative increase in the expression level ofone or more promoters (i.e., miR-199a-3p, miR-199a-5p, miR-1908, andCTGF) is indicative of presence the disorder. Conversely, a lowerexpression level or a lack of the expression is indicative lack of thedisorder.

The presence, level, or absence of, an mRNA, microRNA, or polypeptideproduct in a test sample can be evaluated by obtaining a test samplefrom a test subject and contacting the test sample with a compound or anagent capable of detecting the nucleic acid (e.g., RNA or DNA probe) orpolypeptide. The “test sample” includes tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. The level of expression of a gene(s) ofinterest can be measured in a number of ways, including measuring theRNA encoded by the gene.

Expressed RNA samples can be isolated from biological samples using anyof a number of well-known procedures. For example, biological samplescan be lysed in a guanidinium-based lysis buffer, optionally containingadditional components to stabilize the RNA. In some embodiments, thelysis buffer can contain purified RNAs as controls to monitor recoveryand stability of RNA from cell cultures. Examples of such purified RNAtemplates include the Kanamycin Positive Control RNA from PROMEGA(Madison, Wis.), and 7.5 kb Poly(A)-Tailed RNA from LIFE TECHNOLOGIES(Rockville, Md.). Lysates may be used immediately or stored frozen at,e.g., −80° C.

Optionally, total RNA can be purified from cell lysates (or other typesof samples) using silica-based isolation in an automation-compatible,96-well format, such as the RNEASY purification platform (QIAGEN, Inc.,Valencia, Calif.). Other RNA isolation methods are contemplated, such asextraction with silica-coated beads or guanidinium. Further methods forRNA isolation and preparation can be devised by one skilled in the art.

The methods of the present invention can be performed using crudesamples (e.g., blood, serum, plasma, or cell lysates), eliminating theneed to isolate RNA. RNAse inhibitors are optionally added to the crudesamples. When using crude cellular lysates, it should be noted thatgenomic DNA can contribute one or more copies of a target sequence,e.g., a gene, depending on the sample. In situations in which the targetsequence is derived from one or more highly expressed genes, the signalarising from genomic DNA may not be significant. But for genes expressedat low levels, the background can be eliminated by treating the sampleswith DNAse, or by using primers that target splice junctions forsubsequent priming of cDNA or amplification products.

The level of RNA corresponding to a gene in a cell can be determinedboth in situ and in vitro. RNA isolated from a test sample can be usedin hybridization or amplification assays that include, Southern orNorthern analyses, PCR analyses, and probe arrays. A preferreddiagnostic method for the detection of RNA levels involves contactingthe isolated RNA with a nucleic acid probe that can hybridize to the RNAencoded by the gene. The probe can be a full-length nucleic acid or aportion thereof, such as an oligonucleotide of at least 10 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to the RNA.

In one format, RNA (or cDNA prepared from it) is immobilized on asurface and contacted with the probes, for example, by running theisolated RNA on an agarose gel and transferring the RNA from the gel toa membrane, such as nitrocellulose. In another format, the probes areimmobilized on a surface and the RNA (or cDNA) is contacted with theprobes, for example, in a gene chip array. A skilled artisan can adaptknown RNA detection methods for detecting the level of RNA.

The level of RNA (or cDNA prepared from it) in a sample encoded by agene to be examined can be evaluated with nucleic acid amplification,e.g., by standard PCR (U.S. Pat. No. 4,683,202), RT-PCR (Bustin S. J MolEndocrinol. 25:169-93, 2000), quantitative PCR (Ong Y. et al.,Hematology. 7:59-67, 2002), real time PCR (Ginzinger D. Exp Hematol.30:503-12, 2002), and in situ PCR (Thaker V. Methods Mol Biol.115:379-402, 1999), or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. In another embodiment, the methods of the inventionfurther include contacting a control sample with a compound or agentcapable of detecting the RNA of a gene and comparing the presence of theRNA in the control sample with the presence of the RNA in the testsample.

The above-described methods and markers can be used to assess the riskof a subject for developing melanoma. In particular, the invention canbe applied to those in high risk cohort who already have certain risksso as to gain critical insight into early detection. A change in levelsof miR gene products associated with melanoma can be detected prior to,or in the early stages of, the development of transformed or neoplasticphenotypes in cells of a subject. The invention therefore also providesa method for screening a subject who is at risk of developing melanoma,comprising evaluating the level of at least one gene product, or acombination of gene products, associated with melanoma in a biologicalsample obtained form the subject's skin. Accordingly, an alteration inthe level of the gene product, or combination of gene products, in thebiological sample as compared to the level of a corresponding geneproduct in a control sample, is indicative of the subject being at riskfor developing melanoma. The biological sample used for such screeningcan include skin tissue that is either normal or suspected to becancerous. Subjects with a change in the level of one or more geneproducts associated with melanoma are candidates for further monitoringand testing. Such further testing can comprise histological examinationof tissue samples, or other techniques within the skill in the art.

As used herein, the term “diagnosis” means detecting a disease ordisorder or determining the stage or degree of a disease or disorder.Usually, a diagnosis of a disease or disorder is based on the evaluationof one or more factors and/or symptoms that are indicative of thedisease. That is, a diagnosis can be made based on the presence, absenceor amount of a factor which is indicative of presence or absence of thedisease or condition. Each factor or symptom that is considered to beindicative for the diagnosis of a particular disease does not need beexclusively related to the particular disease; i.e. there may bedifferential diagnoses that can be inferred from a diagnostic factor orsymptom. Likewise, there may be instances where a factor or symptom thatis indicative of a particular disease is present in an individual thatdoes not have the particular disease. The diagnostic methods may be usedindependently, or in combination with other diagnosing and/or stagingmethods known in the medical art for a particular disease or disorder,e.g., melanoma.

Prognosis Methods

The diagnostic methods described above can identify subjects having, orat risk of developing, a melanoma. In addition, changes in expressionlevels and/or trends of the above-mentioned genes in a biologicalsample, e.g., peripheral blood samples, can provide an early indicationof recovery or lack thereof. For example, a further increase (ordecline) or persistently-altered gene expression levels of the promotergenes (or inhibitor genes) indicate a poor prognosis, i.e., lack ofimprovement or health decline. Accordingly, these genes allow one toassess post-treatment recovery of melanoma. The analysis of this selectgroup of genes or a subset thereof indicates outcomes of the conditions.

The prognostic assays described herein can be used to determine whethera subject is suitable to be administered with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat melanoma or otherdisorders associated with endothelial recruitment, cancer cell invasion,or metastatic angiogenesis. For example, such assays can be used todetermine whether a subject can be administered with a chemotherapeuticagent.

Thus, also provided by this invention is a method of monitoring atreatment for a cellular proliferative disorder in a subject. For thispurpose, gene expression levels of the genes disclosed herein can bedetermined for test samples from a subject before, during, or afterundergoing a treatment. The magnitudes of the changes in the levels ascompared to a baseline level are then assessed. A decrease in theexpression of the above-mentioned promoter genes (miR-199a-3p,miR-199a-5p, miR-1908, and CTGF) after the treatment indicates that thesubject can be further treated by the same treatment. Similarly, anincrease in the inhibitors (DNAJA4, ApoE, LRP1, and LRP8) also indicatesthat the subject can be further treated by the same treatment.Conversely, further increase or persistent high expression levels of oneor more of the promoter genes is indicate lack of improvement or healthdecline.

Information obtained from practice of the above assays is useful inprognostication, identifying progression of, and clinical management ofdiseases and other deleterious conditions affecting an individualsubject's health status. In preferred embodiments, the foregoingdiagnostic assays provide information useful in prognostication,identifying progression of and management of melanoma and otherconditions characterized by endothelial recruitment, cancer cellinvasion, or metastatic angiogenesis. The information more specificallyassists the clinician in designing chemotherapeutic or other treatmentregimes to eradicate such conditions from the body of an afflictedsubject, a human.

The term “prognosis” as used herein refers to a prediction of theprobable course and outcome of a clinical condition or disease. Aprognosis is usually made by evaluating factors or symptoms of a diseasethat are indicative of a favorable or unfavorable course or outcome ofthe disease. The phrase “determining the prognosis” as used hereinrefers to the process by which the skilled artisan can predict thecourse or outcome of a condition in a patient. The term “prognosis” doesnot refer to the ability to predict the course or outcome of a conditionwith 100% accuracy instead, the skilled artisan will understand that theterm “prognosis” refers to an increased probability that a certaincourse or outcome will occur; that is, that a course or outcome is morelikely to occur in a patient exhibiting a given condition, when comparedto those individuals not exhibiting the condition.

The terms “favorable prognosis” and “positive prognosis,” or“unfavorable prognosis” and “negative prognosis” as used herein arerelative terms for the prediction of the probable course and/or likelyoutcome of a condition or a disease. A favorable or positive prognosispredicts a better outcome for a condition than an unfavorable ornegative prognosis. In a general sense, a “favorable prognosis” is anoutcome that is relatively better than many other possible prognosesthat could be associated with a particular condition, whereas anunfavorable prognosis predicts an outcome that is relatively worse thanmany other possible prognoses that could be associated with a particularcondition. Typical examples of a favorable or positive prognosis includea better than average cure rate, a lower propensity for metastasis, alonger than expected life expectancy, differentiation of a benignprocess from a cancerous process, and the like. For example, a positiveprognosis is one where a patient has a 50% probability of being cured ofa particular cancer after treatment, while the average patient with thesame cancer has only a 25% probability of being cured.

The terms “determining,” “measuring,” “assessing,” and “assaying” areused interchangeably and include both quantitative and qualitativemeasurement, and include determining if a characteristic, trait, orfeature is present or not. Assessing may be relative or absolute.“Assessing the presence of” a target includes determining the amount ofthe target present, as well as determining whether it is present orabsent.

Arrays

Also provided in the invention is a biochip or array. The biochip/arraymay contain a solid or semi-solid substrate having an attached probe orplurality of probes described herein. The probes may be capable ofhybridizing to a target sequence under stringent hybridizationconditions. The probes may be attached at spatially defined address onthe substrate. More than one probe per target sequence may be used, witheither overlapping probes or probes to different sections of aparticular target sequence. The probes may be capable of hybridizing totarget sequences associated with a single disorder appreciated by thosein the art. The probes may either be synthesized first, with subsequentattachment to the biochip, or may be directly synthesized on thebiochip.

“Attached” or “immobilized” as used herein to refer to a nucleic acid(e.g., a probe) and a solid support may mean that the binding betweenthe probe and the solid support is sufficient to be stable underconditions of binding, washing, analysis, and removal. The binding maybe covalent or non-covalent. Covalent bonds may be formed directlybetween the probe and the solid support or may be formed by a crosslinker or by inclusion of a specific reactive group on either the solidsupport or the probe or both molecules. Non-covalent binding may be oneor more of electrostatic, hydrophilic, and hydrophobic interactions.Included in non-covalent binding is the covalent attachment of amolecule, such as streptavidin, to the support and the non-covalentbinding of a biotinylated probe to the streptavidin. Immobilization mayalso involve a combination of covalent and non-covalent interactions.

The solid substrate can be a material that may be modified to containdiscrete individual sites appropriate for the attachment or associationof the probes and is amenable to at least one detection method. Examplesof such substrates include glass and modified or functionalized glass,plastics (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses and plastics. The substratesmay allow optical detection without appreciably fluorescing.

The substrate can be planar, although other configurations of substratesmay be used as well. For example, probes may be placed on the insidesurface of a tube, for flow-through sample analysis to minimize samplevolume. Similarly, the substrate may be flexible, such as flexible foam,including closed cell foams made of particular plastics.

The array/biochip and the probe may be derivatized with chemicalfunctional groups for subsequent attachment of the two. For example, thebiochip may be derivatized with a chemical functional group including,but not limited to, amino groups, carboxyl groups, oxo groups or thiolgroups. Using these functional groups, the probes may be attached usingfunctional groups on the probes either directly or indirectly using alinker. The probes may be attached to the solid support by either the 5′terminus, 3′ terminus, or via an internal nucleotide. The probe may alsobe attached to the solid support non-covalently. For example,biotinylated oligonucleotides can be made, which may bind to surfacescovalently coated with streptavidin, resulting in attachment.Alternatively, probes may be synthesized on the surface using techniquessuch as photopolymerization and photolithography. Detailed discussion ofmethods for linking nucleic acids to a support substrate can be foundin, e.g., U.S. Pat. Nos. 5,837,832, 6,087,112, 5,215,882, 5,707,807,5,807,522, 5,958,342, 5,994,076, 6,004,755, 6,048,695, 6,060,240,6,090,556, and 6,040,138.

In some embodiments, an expressed transcript (e.g., a transcript of amicroRNA gene described herein) is represented in the nucleic acidarrays. In such embodiments, a set of binding sites can include probeswith different nucleic acids that are complementary to differentsequence segments of the expressed transcript. Examples of such nucleicacids can be of length of 15 to 200 bases, 20 to 100 bases, 25 to 50bases, 40 to 60 bases. Each probe sequence can also include one or morelinker sequences in addition to the sequence that is complementary toits target sequence. A linker sequence is a sequence between thesequence that is complementary to its target sequence and the surface ofsupport. For example, the nucleic acid arrays of the invention can haveone probe specific to each target microRNA gene. However, if desired,the nucleic acid arrays can contain at least 2, 5, 10, 100, 200, 300,400, 500 or more probes specific to some expressed transcript (e.g., atranscript of a microRNA gene described herein).

Kits

In another aspect, the present invention provides kits embodying themethods, compositions, and systems for analysis of the polypeptides andmicroRNA expression as described herein. Such a kit may contain anucleic acid described herein together with any or all of the following:assay reagents, buffers, probes and/or primers, and sterile saline oranother pharmaceutically acceptable emulsion and suspension base. Inaddition, the kit may include instructional materials containingdirections (e.g., protocols) for the practice of the methods describedherein. For example, the kit may be a kit for the amplification,detection, identification or quantification of a target mRNA or microRNAsequence. To that end, the kit may contain a suitable primer (e.g.,hairpin primers), a forward primer, a reverse primer, and a probe.

In one example, a kit of the invention includes one or more microarrayslides (or alternative microarray format) onto which a plurality ofdifferent nucleic acids (each corresponding to one of theabove-mentioned genes) have been deposited. The kit can also include aplurality of labeled probes. Alternatively, the kit can include aplurality of polynucleotide sequences suitable as probes and a selectionof labels suitable for customizing the included polynucleotidesequences, or other polynucleotide sequences at the discretion of thepractitioner. Commonly, at least one included polynucleotide sequencecorresponds to a control sequence, e.g., a normalization gene or thelike. Exemplary labels include, but are not limited to, a fluorophore, adye, a radiolabel, an enzyme tag, that is linked to a nucleic acidprimer.

In one embodiment, kits that are suitable for amplifying nucleic acidcorresponding to the expressed RNA samples are provided. Such a kitincludes reagents and primers suitable for use in any of theamplification methods described above. Alternatively, or additionally,the kits are suitable for amplifying a signal corresponding tohybridization between a probe and a target nucleic acid sample (e.g.,deposited on a microarray).

In addition, one or more materials and/or reagents required forpreparing a biological sample for gene expression analysis areoptionally included in the kit. Furthermore, optionally included in thekits are one or more enzymes suitable for amplifying nucleic acids,including various polymerases (RT, Taq, etc.), one or moredeoxynucleotides, and buffers to provide the necessary reaction mixturefor amplification.

Typically, the kits are employed for analyzing gene expression patternsusing mRNA or microRNA as the starting template. The RNA template may bepresented as either total cellular RNA or isolated RNA; both types ofsample yield comparable results. In other embodiments, the methods andkits described in the present invention allow quantitation of otherproducts of gene expression, including tRNA, rRNA, or othertranscription products.

Optionally, the kits of the invention further include software toexpedite the generation, analysis and/or storage of data, and tofacilitate access to databases. The software includes logicalinstructions, instructions sets, or suitable computer programs that canbe used in the collection, storage and/or analysis of the data.Comparative and relational analysis of the data is possible using thesoftware provided.

The kits optionally contain distinct containers for each individualreagent and/or enzyme component. Each component will generally besuitable as aliquoted in its respective container. The container of thekits optionally includes at least one vial, ampule, or test tube.Flasks, bottles and other container mechanisms into which the reagentscan be placed and/or aliquoted are also possible. The individualcontainers of the kit are preferably maintained in close confinement forcommercial sale. Suitable larger containers may include injection orblow-molded plastic containers into which the desired vials areretained. Instructions, such as written directions or videotapeddemonstrations detailing the use of the kits of the present invention,are optionally provided with the kit.

In a further aspect, the present invention provides for the use of anycomposition or kit herein, for the practice of any method or assayherein, and/or for the use of any apparatus or kit to practice any assayor method herein.

A “test sample” or a “biological sample” as used herein may mean asample of biological tissue or fluid that comprises nucleic acids. Suchsamples include, but are not limited to, tissue or body fluid isolatedfrom animals. Biological samples may also include sections of tissuessuch as biopsy and autopsy samples, frozen sections taken forhistological purposes, blood, plasma, serum, sputum, stool, tears,mucus, urine, effusions, amniotic fluid, ascitic fluid, hair, and skin.Biological samples also include explants and primary and/or transformedcell cultures derived from patient tissues. A biological sample may beprovided by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods described herein in vivo. Archival tissues, suchas those having treatment or outcome history, may also be used.

The term “body fluid” or “bodily fluid” refers to any fluid from thebody of an animal. Examples of body fluids include, but are not limitedto, plasma, serum, blood, lymphatic fluid, cerebrospinal fluid, synovialfluid, urine, saliva, mucous, phlegm and sputum. A body fluid sample maybe collected by any suitable method. The body fluid sample may be usedimmediately or may be stored for later use. Any suitable storage methodknown in the art may be used to store the body fluid sample: forexample, the sample may be frozen at about −20° C. to about −70° C.Suitable body fluids are acellular fluids. “Acellular” fluids includebody fluid samples in which cells are absent or are present in such lowamounts that the miRNA level determined reflects its level in the liquidportion of the sample, rather than in the cellular portion. Suchacellular body fluids are generally produced by processing acell-containing body fluid by, for example, centrifugation orfiltration, to remove the cells. Typically, an acellular body fluidcontains no intact cells however, some may contain cell fragments orcellular debris. Examples of acellular fluids include plasma or serum,or body fluids from which cells have been removed.

The term “gene” used herein refers to a natural (e.g., genomic) orsynthetic gene comprising transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (e.g., introns, 5′- and 3′-untranslated sequences). The codingregion of a gene may be a nucleotide sequence coding for an amino acidsequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA,miRNA or antisense RNA. A gene may also be an mRNA or cDNA correspondingto the coding regions (e.g., exons and miRNA) optionally comprising 5′-or 3′-untranslated sequences linked thereto. A gene may also be anamplified nucleic acid molecule produced in vitro comprising all or apart of the coding region and/or 5′- or 3′-untranslated sequences linkedthereto. The term also includes pseudogenes, which are dysfunctionalrelatives of known genes that have lost their protein-coding ability orare otherwise no longer expressed in a cell.

“Expression profile” as used herein refers to a genomic expressionprofile, e.g., an expression profile of microRNAs. Profiles may begenerated by any convenient means for determining a level of a nucleicacid sequence e.g., quantitative hybridization of microRNA, cRNA, etc.,quantitative PCR, ELISA for quantification, and the like, and allow theanalysis of differential gene expression between two samples. A subjector patient sample, e.g., cells or a collection thereof, e.g., tissues,is assayed. Samples are collected by any convenient method, as known inthe art. Nucleic acid sequences of interest are nucleic acid sequencesthat are found to be predictive, including the nucleic acid sequences ofthose described herein, where the expression profile may includeexpression data for 5, 10, 20, 25, 50, 100 or more of, including all ofthe listed nucleic acid sequences. The term “expression profile” mayalso mean measuring the abundance of the nucleic acid sequences in themeasured samples.

“Differential expression” refers to qualitative or quantitativedifferences in the temporal and/or cellular gene expression patternswithin and among cells and tissue. Thus, a differentially expressed genecan qualitatively have its expression altered, including an activationor inactivation, in, e.g., normal versus disease tissue. Genes may beturned on or turned off in a particular state, relative to another statethus permitting comparison of two or more states. A qualitativelyregulated gene will exhibit an expression pattern within a state or celltype that may be detectable by standard techniques. Some genes will beexpressed in one state or cell type, but not in both. Alternatively, thedifference in expression may be quantitative, e.g., in that expressionis modulated, up-regulated, resulting in an increased amount oftranscript, or down-regulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques such asexpression arrays, quantitative reverse transcriptase PCR, Northernanalysis, and RNase protection.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinrefers to at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

The term “primer” refers to any nucleic acid that is capable ofhybridizing at its 3′ end to a complementary nucleic acid molecule, andthat provides a free 3′ hydroxyl terminus which can be extended by anucleic acid polymerase. As used herein, amplification primers are apair of nucleic acid molecules that can anneal to 5′ or 3′ regions of agene (plus and minus strands, respectively, or vice-versa) and contain ashort region in between. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule having the nucleotide sequence flanked by the primers. Forin situ methods, a cell or tissue sample can be prepared and immobilizedon a support, such as a glass slide, and then contacted with a probethat can hybridize to RNA. Alternative methods for amplifying nucleicacids corresponding to expressed RNA samples include those described in,e.g., U.S. Pat. No. 7,897,750.

The term “probe” as used herein refers to an oligonucleotide capable ofbinding to a target nucleic acid of complementary sequence through oneor more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. Probes may bind targetsequences lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridization conditions. There maybe any number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids described herein. However, if the number of mutations isso great that no hybridization can occur under even the least stringentof hybridization conditions, the sequence is not a complementary targetsequence. A probe may be single stranded or partially single andpartially double stranded. The strandedness of the probe is dictated bythe structure, composition, and properties of the target sequence.Probes may be directly labeled or indirectly labeled such as with biotinto which a streptavidin complex may later bind.

“Complement” or “complementary” as used herein to refer to a nucleicacid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen basepairing between nucleotides or nucleotide analogs of nucleic acidmolecules. A full complement or fully complementary may mean 100%complementary base pairing between nucleotides or nucleotide analogs ofnucleic acid molecules.

“Stringent hybridization conditions” as used herein refers to conditionsunder which a first nucleic acid sequence (e.g., probe) hybridizes to asecond nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand be different in different circumstances, and can be suitablyselected by one skilled in the art. Stringent conditions may be selectedto be about 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. The Tm may be thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. However, severalfactors other than temperature, such as salt concentration, caninfluence the stringency of hybridization and one skilled in the art cansuitably select the factors to accomplish a similar stringency.

As used herein the term “reference value” refers to a value thatstatistically correlates to a particular outcome when compared to anassay result. In preferred embodiments, the reference value isdetermined from statistical analysis of studies that compare microRNAexpression with known clinical outcomes. The reference value may be athreshold score value or a cutoff score value. Typically a referencevalue will be a threshold above (or below) which one outcome is moreprobable and below which an alternative threshold is more probable.

In one embodiment, a reference level may be one or more circulatingmiRNA levels expressed as an average of the level of the circulatingmiRNA from samples taken from a control population of healthy(disease-free) subjects. In another embodiment, the reference level maybe the level in the same subject at a different time, e.g., before thepresent assay, such as the level determined prior to the subjectdeveloping the disease or prior to initiating therapy. In general,samples are normalized by a common factor. For example, acellular bodyfluid samples are normalized by volume body fluid and cell-containingsamples are normalized by protein content or cell count. Nucleic acidsamples may also be normalized relative to an internal control nucleicacid.

As disclosed herein, the difference of the level of one or morepolypeptides or RNAs (mRNAs or microRNAs) is indicative of a disease ora stage thereof. The phrase “difference of the level” refers todifferences in the quantity of a particular marker, such as a nucleicacid, in a sample as compared to a control or reference level. Forexample, the quantity of a particular biomarker may be present at anelevated amount or at a decreased amount in samples of patients with aneoplastic disease compared to a reference level. In one embodiment, a“difference of a level” may be a difference between the quantity of aparticular biomarker present in a sample as compared to a control (e.g.,reference value) of at least about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 60%, 75%, 80% 100%, 150%, 200%, or more. In oneembodiment, a “difference of a level” may be a statistically significantdifference between the quantities of a biomarker present in a sample ascompared to a control. For example, a difference may be statisticallysignificant if the measured level of the biomarker falls outside ofabout 1.0 standard deviation, about 1.5 standard deviations, about 2.0standard deviations, or about 2.5 stand deviations of the mean of anycontrol or reference group. With respect to miRNA measurement, the levelmay be measured from real-time PCR as the Ct value, which may benormalized to a ΔCt value as described in the Examples below.

Drug Screening

The invention provides a method for identifying a compound that areuseful for treating melanoma or for inhibiting endothelial recruitment,cell invasion, or metastatic angiogenesis.

Candidate compounds to be screened (e.g., proteins, peptides,peptidomimetics, peptoids, antibodies, small molecules, or other drugs)can be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art. Such libraries include: peptidelibraries, peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbone thatis resistant to enzymatic degradation); spatially addressable parallelsolid phase or solution phase libraries; synthetic libraries obtained bydeconvolution or affinity chromatography selection; and the “one-beadone-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med.Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. Examplesof methods for the synthesis of molecular libraries can be found in,e.g., DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNAS USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994 J. Med. Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten, 1992, Biotechniques13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor,1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores(U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, PNAS USA89:1865-1869), or phages (Scott and Smith 1990, Science 249:386-390;Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNAS USA87:6378-6382; Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Pat. No.5,223,409).

To identify a useful compound, one can contact a test compound with asystem containing test cells expressing a reporter gene encoded by anucleic acid operatively liked to a promoter of a marker gene selectedfrom the above-mentioned metastasis promoters or suppressors. The systemcan be an in vitro cell line model or an in vivo animal model. The cellscan naturally express the gene, or can be modified to express arecombinant nucleic acid. The recombinant nucleic acid can contain anucleic acid coding a reporter polypeptide to a heterologous promoter.One then measures the expression level of the miRNA, polypeptide, orreporter polypeptide.

For the polypeptide, the expression level can be determined at eitherthe mRNA level or at the protein level. Methods of measuring mRNA levelsin a cell, a tissue sample, or a body fluid are well known in the art.To measure mRNA levels, cells can be lysed and the levels of mRNA in thelysates or in RNA purified or semi-purified from the lysates can bedetermined by, e.g., hybridization assays (using detectably labeledgene-specific DNA or RNA probes) and quantitative or semi-quantitativeRT-PCR (using appropriate gene-specific primers). Alternatively,quantitative or semi-quantitative in situ hybridization assays can becarried out using tissue sections or unlysed cell suspensions, anddetectably (e.g., fluorescent or enzyme) labeled DNA or RNA probes.Additional mRNA-quantifying methods include RNA protection assay (RPA)and SAGE. Methods of measuring protein levels in a cell or a tissuesample are also known in the art.

To determine the effectiveness of a candidate compound to treat melanomaor inhibiting endothelial recruitment, cell invasion, or metastaticangiogenesis, one can compare the level obtained in the manner describedabove with a control level (e.g., one obtained in the absence of thecandidate compound). The compound is identified as being effective if(i) a metastasis suppressor's level is lower than a control or referencevalue or (ii) a metastasis promoter's level is higher than the controlor reference value. One can further verify the efficacy of a compoundthus-identified using the in vitro cell culture model or an in vivoanimal model as disclosed in the example below.

EXAMPLES Example 1 Materials And Methods

This example describes materials and methods used in EXAMPLES 2-11below.

Compounds

TABLE 3 Compound Names Compound # Compound Name 1 T0901317 2 GW3965 3LXR-623 12 WO-2010-0138598 Ex. 9 or WO-201000138598 25 SB742881 38WO-2007-002563 Ex. 19 or WO-2007-002563

Animal Studies

All mouse experiments were conducted in agreement with a protocolapproved by the Institutional Animal Care and Use Committee (IACUC) atThe Rockefeller University. 6-8-week old age-matched and sex-matchedmice were used for primary tumor growth and metastasis assays aspreviously described (Minn et al., 2005; Tavazoie et al., 2008). SeeExtended Experimental Procedures.

Cell Culture

All cancer cell lines were cultured as previously described (Tavazoie etal., 2008). 293T and human umbilical vein endothelial cells (HUVEC's)were maintained in standard conditions. miRNA and geneknock-down/over-expression studies in cell lines and in vitro functionalassays are detailed in Extended Experimental Procedures.

Microarray Hybridization

In order to identify miRNAs deregulated across highly metastaticderivatives, small RNAs were enriched from total RNA derived from MeWoand A375 cell lines and profiled by LC sciences. In order to identifypotential gene targets of miR-199a-3p, miR-199a-5p, and miR-1908, totalRNA from MeWo cell lines was labeled and hybridized onto Illumina HT-12v3 Expression BeadChip arrays by The Rockefeller University genomicscore facility. See Extended Experimental Procedures for thresholds andcriteria used to arrive at miRNA and mRNA targets.

Analysis of miRNA Expression in Human Melanoma Skin Lesions

All human clinical samples used in this study were obtained, processed,and analyzed in accordance with IRB guidelines. Total RNA was extractedfrom paraffin-embedded cross-sections of primary melanoma skin lesionspreviously resected from patients at MSKCC, and specific miRNAexpression levels were analyzed in a blinded fashion using TaqMan miRNAAssays (Applied Biosystems). Kaplan-Meier curves representing eachpatient's metastasis-free-survival data as a function of primary tumormiRNA expression values were generated using the GraphPad Prism softwarepackage.

In Vivo LNA Therapy

Following tail-vein injection of 4×10⁴ MeWo-LM2 cells, NOD-SCID micewere treated intravenously twice a week for four weeks with invivo-optimized LNAs (Exiqon) antisense to miR-199a-3p, miR-199a-5p, andmiR-1908 at a combinatorial dose of 12.5 mg/kg delivered in 0.1 mL ofPBS.

Histology

For gross macroscopic metastatic nodule visualization, 5-μm-thick lungtissue sections were H&E stained. For in vivo endothelial contentanalyses, lung sections were double-stained with antibodies againstMECA-32 (Developmental Studies Hybridoma Bank, The University of Iowa,IA), which labels mouse endothelial cells, and human vimentin (VectorLaboratories), which labels human melanoma cells. See ExtendedExperimental Procedures.

Data Analysis

All data are represented as mean±SEM. The Kolmogorov-Smirnov test wasused to determine significance of differences in metastatic blood vesseldensity cumulative distributions. The prognostic power of the miRNAs topredict metastatic outcomes was tested for significance using theMantel-Cox log-rank test. The one-way Mann-Whitney t-test was used todetermine significance values for non-Gaussian bioluminescencemeasurements. For all other comparisons, the one-sided student's t-testwas used. P values <0.05 were deemed to be statistically significant.

In Vivo Selection, Experimental Metastasis, and Primary Tumor GrowthAssays

All mouse experiments were conducted in agreement with a protocolapproved by the Institutional Animal Care and Use Committee (IACUC) atThe Rockefeller University. To generate multiple metastatic derivativesfrom two independent human melanoma cell lines, in vivo selection wasperformed as previously described (Minn et al., 2005 Nature 436,518-524; Pollack and Fidler, 1982 J. Natl. Cancer Inst. 69, 137-141). Inbrief, 1×10⁶ pigmented MeWo or non-pigmented A375 melanoma parentalcells were resuspended in 0.1 mL of PBS and intravenously injected into6-8-week old immunocompromised NOD-SCID mice. Following lung metastasesformation, nodules were dissociated and cells were propagated in vitro,giving rise to first generation of lung metastatic derivatives (LM1).The LM1 cells were then subjected to another round of in vivo selectionby injecting 2×10⁵ cells via the tail-vein into NOD-SCID mice, givingrise to metastatic nodules, whose subsequent dissociation yielded secondgeneration of lung metastatic derivatives (LM2). For the A375 cell line,a third round of in vivo selection was performed, yielding the highlymetastatic A375-LM3 derivatives.

In order to monitor metastasis in vivo through bioluminescence imaging,A375 and MeWo parental cells and their metastatic derivatives weretransduced with a retroviral construct expressing a luciferase reporter(Ponomarev et al., 2004 Eur J Nucl Med Mol Imaging 31, 740-751). For allmetastasis experiments, lung or systemic colonization was monitored overtime and quantified through non-invasive bioluminescence imaging aspreviously described (Minn et al., 2005). To determine whether in vivoselection had been achieved, 4×10⁴ MeWo parental or MeWo-LM2 cells and1×10⁵ A375 parental or A375-LM3 cells were resuspended in 0.1 mL of PBSand injected via the lateral tail vein into 6-8-week old NOD-SCID mice.For experimental metastasis assays testing the effects of putativepromoter miRNAs on lung colonization, 4×10⁴ MeWo parental cellsover-expressing miR-199a, miR-1908, miR-214, or a control hairpin, 4×10⁴MeWo-LM2 cells with silenced expression of miR-199a-3p, miR-199a-5p,miR-1908, or a control sequence, and 2×10⁵ A375-LM3 cells inhibited formiR-199a-3p, miR-199a-5p, miR-1908, or a control sequence wereresuspended in 0.1 mL of PBS and tail-vein injected into 6-8-week oldNOD-SCID mice. For epistasis experiments, 1×10⁵ MeWo-LM2 cellsexpressing an shRNA targeting ApoE, DNAJA4, or a control sequence orsiRNA inhibiting LRP1 or a control sequence in the setting of miRNAinhibition were intravenously injected into 6-8-week old NOD-SCID mice.For ApoE pre-treatment experiments, MeWo-LM2 cells were incubated in thepresence of ApoE or BSA at 100 μg/mL at 37° C. After 24 hours, 4×10⁴cells were injected via the tail-vein into 7-week old NOD-SCID mice. Todetermine the effect of pre-treating highly metastatic melanoma cellswith LNAs targeting miR-199a-3p, miR-199a-5p, and miR-1908 onmetastasis, MeWo-LM2 cells were transfected with each LNA individually,a cocktail of LNAs targeting all three miRNAs, or a control LNA. After48 hours, 1×10⁵ cells, resuspended in 0.1 mL of PBS, were administeredintravenously into 7-week old NOD-SCID mice for lung metastaticcolonization studies or through intracardiac injection into 7-week oldathymic nude mice for systemic metastasis assays. To determine theeffect of genetic deletion of ApoE on metastasis, 8-week old C57BL/6-WTor C57BL/6-ApoE−/− mice were intravenously injected with 5×10⁴ B16F10mouse melanoma cells. For primary tumor growth studies, 1×10⁶ parentalMeWo cells over-expressing miR-199a, miR-1908, or a control hairpin weremixed 1:1 with matrigel and subcutaneously injected into the lower rightflank of 6-week old immunodeficient NOD-SCID mice. Animals were palpatedweekly for tumor formation, after which sizeable tumors were measuredtwice a week. Tumor volume was calculated as (small diameter) x (largediameter)/2.

Lentiviral miRNA Inhibition and Gene Knock-Down

293T cells were seeded in a 10-cm plate and allowed to reach 60%confluency. Prior to transfection, the cell media was replaced withfresh antibiotic-free DMEM media supplemented with 10% FBS. 6 μg ofvector A, 12 μg of vector K, and 12 μg of the appropriate miR-Zip(System Biosciences, Mountain View, Calif.) or shRNA plasmid construct(MSKCC HTS Core Facility, New York, N.Y.) were co-transfected using 60μL of TransIT-293 transfection reagent (MIR 2700, Minis Bio LLC,Madison, Wis.). The cells were incubated at 37° C. for 48 hours, and thevirus was harvested by spinning the cell media for 10 minutes at 2000 gfollowed by virus filtration through a 0.45 μm filter. 1×10⁵ cancercells were transduced with 2 mL of the appropriate virus in the presenceof 10 μg/mL of polybrene (TR-1003-G, Millipore, Billerica, Mass.) for 6hrs. After 48 hours, 2 μg/mL of puromycin (P8833, Sigma-Aldrich, StLouis, Mo.) was added to the cell media for lentiviral selection. Thecells were kept in puromycin selection for 72 hours. The followingmiR-Zip sequences were used:

miR-Zip-199a-3p: 5′-GATCCGACAGTAGCCTGCACATTAGTCACTTCCTGTCAGTAACCAATGTGCAGACTACTGTTTTTTGAATT-3′ miR-Zip-199a-5p:5′-GATCCGCCCAGTGCTCAGACTACCCGTGCCTTCCTGTCAGGAACAGGTAGTCTGAACACTGGGTTTTTGAATT-3′ miR-Zip-19085′-GATCCGCGGCGGGAACGGCGATCGGCCCTTCCTGTCAGGACCAATCGCCGTCCCCGCCGTTTTTGAATT-3′

The following shRNA sequences were used:

shAPOE¹: 5′CCGGGAAGGAGTTGAAGGCCTACAACTCGAGTTGTAGGCCTTCAACTCC TTCTTTTT3′shAPOE²: 5′CCGGGCAGACACTGTCTGAGCAGGTCTCGAGACCTGCTCAGACAGTGTC TGCTTTTT3′shDNAJA4¹: 5′CCGGGCGAGAAGTTTAAACTCATATCTCGAGATATGAGTTTAAACTTCTCGCTTTTT3′ shDNAJA4²:5′CCGGCCTCGACAGAAAGTGAGGATTCTCGAGAATCCTCACTTTCTGTCG AGGTTTTT3′Retroviral miRNA and Gene Over-Expression

6 μg of vector VSVG, 12 μg of vector Gag-Pol, and 12 μg of pBabe plasmidcontaining the coding sequences of human ApoE, DNAJA4, or an emptyvector or miR-Vec containing the precursor sequence of miR-199a,miR-214, miR-1908, or a control hairpin were co-transfected into60%-confluent 293T cells using 60 μL of TransIT-293 transfectionreagent. The cells were incubated at 37° C. for 48 hours, after whichthe virus was harvested and transduced into cancer cells in the presenceof 10 μg/mL of polybrene for 6 hours. After 48 hours, 2 μg/mL ofpuromycin or 10 μg/mL of blasticidin (15205, Sigma-Aldrich, St Louis,Mo.) were added to the cell media for retroviral selection. The cellswere kept in puromycin selection for 72 hours or in blasticidinselection for 7 days. The following cloning primers were used forover-expression of the coding sequences of ApoE and DNAJA4:

ApoE_CDS_Fwd: 5′-TCATGAGGATCCATGAAGGTTCTGTGGGCT-3′ ApoE_CDS_Rev:5′-TAGCAGAATTCTCAGTGATTGTCGCTGGG-3′ DNAJA4_CDS_Fwd:5′-ATCCCTGGATCCATGTGGGAAAGCCTGACCC-3′ DNAJA4_CDS_Rev:5′-TACCATGTCGACTCATGCCGTCTGGCACTGC-3′LNA-Based miRNA Knock-Down

LNAs complimentary to mature miR-199a-3p, miR-199a-5p, miR-1908, or acontrol sequence (426917-00, 426918-00, 426878-00, and 1990050,respectively; Exiqon, Vedbaek, Denmark) were transfected at a finalconcentration of 50 nM into 50% confluent MeWo-LM2 cancer cells culturedin antibiotics-free media using Lipofectamine™ 2000 transfection reagent(11668-09, Invitrogen, Carlsbad, Calif.). After 8 hours, thetransfection media was replaced with fresh media. After 48 hours, 1×10⁵cells were injected intravenously into NOD-SCID mice to assess lungmetastatic colonization or through intracardiac injection into athymicnude mice to assess systemic metastasis. For cell invasion andendothelial recruitment in vitro assays, the cells were used 96 hourspost-transfection.

siRNA-Based mRNA Knock-Down

siRNAs targeting LRP1, LRP8, VLDLR, LDLR, or a control sequence weretransfected into cancer cells or HUVEC's at a final concentration of 100nM using Lipofectamine™ 2000 transfection reagent. After 5 hours, thetransfection media was replaced with fresh media. The cells weresubjected to matrigel invasion and endothelial recruitment assays 96hours post-transfection. Cells transduced with siRNAs targeting LRP1 ora control sequence in the setting of miRNA inhibition were tail-veininjected for lung colonization assays 72 hours post-transfection.Control non-targeting siRNAs were obtained from Dharmacon. The followingLRP1 and LRP8 target sequences were used:

siLRP1¹: 5′-CGAGGACGAUGACUGCUUA-3′; siLRP1²: 5′-GCUAUGAGUUUAAGAAGUU-3′;siLRP8¹: 5′-CGAGGACGAUGACUGCUUA-3′; siLRP8²: 5′-GAACUAUUCACGCCUCAUC-3′.

Cell Proliferation Assay

To determine the effects of miR-199a or miR-1908 over-expression andcombinatorial LNA-induced miRNA inhibition on cell proliferation,2.5×10⁴ cells were seeded in triplicate in 6-well plates, and viablecells were counted after 5 days. To assess the effects of recombinantApoE addition on melanoma cell or endothelial cell proliferation, 3×10⁴melanoma MeWo-LM2 cells or endothelial cells were incubated in thepresence of ApoE (100 μM) or BSA (100 μM). Viable cells were countedafter 8, 24, 48, 72, and 120 hours.

Matrigel Invasion Assay

Cancer cells were serum-starved in 0.2% FBS DMEM-based media for 12hours. Trans-well invasion chambers (354480, BD Biosciences, Bedford,Mass.) were pre-equilibrated prior to beginning the assay by adding 0.5mL of starvation media to the top and bottom chambers. After 30 minutes,the media in the top chamber was removed, and 0.5 mL of media containing1×10⁵ cancer cells was added into each matrigel-coated trans-well insertand incubated at 37° C. for 24 hours. For neutralization antibody and/orrecombinant protein experiments, antibody/recombinant protein was addedto each well at the start of the assay at the following concentrationsas indicated in the figures: 5-40 μg/mL anti-ApoE 1D7 (Heart Institute,University of Ottawa), 5-40 μg/mL anti-IgG (AB-108-C, R&D Systems,Minneapolis, Minn.), 100 μM recombinant human ApoE3 (4696, BioVision,Mountain View, Calif.), and 100 μM BSA (A2153, Sigma-Aldrich). Uponcompletion of the assay, matrigel-coated inserts were washed with PBS,the cells at the top side of each insert were scraped off, and theinserts were fixed in 4% paraformaldehyde for 15 minutes. The insertswere then cut out and mounted onto slides using VectaShield mountingmedium containing DAPI (H-1000, Vector Laboratories, Burlingame,Calif.). The basal side of each insert was imaged using an invertedfluorescence microscope (Zeiss Axiovert 40 CFL) at 5× magnification,taking three representative images for each insert. The number ofinvaded cells was quantified using ImageJ (NIH).

Endothelial Recruitment Assay

5×10⁴ cancer cells were seeded into 24-well plates approximately 24hours prior to the start of the assay. HUVEC's were grown to 80%confluency and serum starved in EGM-2 media supplemented with 0.2% FBSfor 16 hours. HUVEC's were then pulsed with Cell Tracker Red CMTPX dye(C34552, Invitrogen) for 45 minutes. Meanwhile, cancer cells were washedwith PBS, 0.5 mL of 0.2% FBS EGM-2 media was added to each well, and a3.0 μm HTS Fluoroblock insert (351151, BD Falcon, San Jose, Calif.) wasplaced into each well. 1×10⁵ HUVEC's, resuspended in 0.5 mL ofstarvation media, were seeded into each trans-well insert, and therecruitment assay was allowed to proceed for 16-18 hours at 37° C. Forneutralization antibody and/or recombinant protein experiments,antibody/protein was then added to each well at the appropriateconcentration as indicated in the figures: 40 μg/mL anti-ApoE 1D7, 40μg/mL anti-IgG, 100 μM rhApoE3, and 100 μM BSA. Upon completion of theassay, the inserts were processed and analyzed as described for thematrigel invasion assay above (See Matrigel Invasion Assay).

Endothelial Migration Assay

Serum-starved HUVEC's were pulsed with Cell Tracker Red CMTPX dye for 45minutes and seeded into HTS Fluoroblock trans-well inserts at aconcentration of 1×10⁵ HUVEC's in 0.5 mL starvation media per eachinsert. The assay was allowed to proceed for 16-18 hours at 37° C., andthe inserts were processed and analyzed as described above (See MatrigelInvasion Assay).

Chemotaxis Assay

HUVEC's were serum-starved in 0.2% FBS EGM-2 media for 16 hours andlabeled with Cell Tracker Red CMTPX dye for 45 minutes. Meanwhile, theindicated amounts (1-5 μg) of recombinant human ApoE3 or BSA were mixedwith 250 μL of matrigel (356231, BD Biosciences) and allowed to solidifyat the bottom of a 24-well plate for 30 min. 250 μL of HUVEC EGM-2 mediacontaining 0.2% FBS was then added to each matrigel-coated well, and 3.0μM HTS Fluoroblock inserts were fitted into each well. 1×10⁵ HUVEC's,resuspended in 0.5 mL of starvation media, were seeded into each insertand allowed to migrate along the matrigel gradient for 16-18 hours at37° C. Upon completion of the assay, the inserts were mounted on slidesand analyzed as described above (See Matrigel Invasion Assay).

Endothelial Adhesion Assay

HUVEC's were seeded in 6-well plates and allowed to form monolayers.Cancer cells were serum starved in 0.2% FBS DMEM-based media for 30minutes and pulsed with Cell Tracker Green CMFDA dye (C7025, Invitrogen)for 45 minutes. 2×10⁵ cancer cells, resuspended in 0.5 mL starvationmedia, were seeded onto each endothelial monolayer. The cancer cellswere allowed to adhere to the HUVEC monolayers for 30 minutes at 37° C.The endothelial monolayers were then washed gently with PBS and fixedwith 4% paraformaldehyde for 15 minutes. Each well was then coated withPBS, and 8 images were taken for each endothelial monolayer using aninverted Fluorescence microscope (Zeiss Axiovert 40 CFL) at 10×magnification. The number of cancer cells adhering to HUVEC's wasquantified using ImageJ.

Anoikis Assay

1×10⁶ MeWo cells over-expressing miR-199a, miR-1908, or a controlhairpin were seeded in low adherent plates containing cell mediasupplemented with 0.2% methylcellulose. Following 48 hours insuspension, the numbers of dead and viable cells were counted usingtrypan blue.

Serum Starvation Assay

To determine the effects of miR-199a and miR-1908 on melanoma cell serumstarvation capacity, 1×10⁵ MeWo parental cells over-expressing miR-199a,miR-1908, or a control hairpin were seeded in quadruplicate into 6-wellplates and incubated in 0.2% FBS starvation DMEM-based media for 48hours, after which the number of viable cells was counted using trypanblue. To determine the effect of recombinant ApoE3 addition on thesurvival of melanoma cells or endothelial cells in serum starvationconditions, 3×10⁴ MeWo-LM2 cells or endothelial cells were incubated inthe presence of ApoE3 (100 μM) or BSA (100 μM) in low serum conditions(0.2% FBS). The number of viable cells was counter after 8, 16, and 24hours.

Colony Formation Assay

Fifty MeWo parental cells over-expressing miR-199a, miR-1908, or acontrol hairpin were seeded in quadruplicate into 6-cm plates. After twoweeks, the cells were washed with PBS, fixed with 6% glutaraldehyde, andstained with 0.5% crystal violet. The number of positive-stainingcolonies was counted.

miRNA Microarray Hybridization

For identification of miRNAs showing deregulated expression acrosshighly metastatic melanoma cell line derivatives, total RNA frommultiple independent metastatic derivatives and their respectiveparental MeWo and A375 cell populations was used to enrich for smallRNAs which were then labelled and hybridized onto microfluidic custommicroarray platforms by LC sciences. The arrays were designed to detect894 mature miRNAs corresponding to the miRNA transcripts listed inSanger miRBase Release 13.0. Out of all the probes analyzed, thosecorresponding to 169 miRNAs yielded signal above a background thresholdacross the multiple cell lines analyzed. The raw signal intensities,corresponding to probe hybridization, were median-normalized for eachcell line. A threshold of 2-fold or higher up-regulation ofmedian-normalized expression values were used in order to identifymiRNAs commonly induced in multiple metastatic derivatives for twoindependent human melanoma cell lines.

Microarray-Based Gene Target Prediction for miR-199a and miR-1908

In order to identify potential genes targeted by miR-199a-3p,miR-199a-5p, and miR-1908, total RNA was extracted from MeWo cell lineswith loss- or gain-of-function of each miRNA and submitted to thegenomics core facility at The Rockefeller University for hybridizationonto Illumina HT-12 v3 Expression BeadChip microarrays. The raw signalintensities, corresponding to probe hybridization, were thenmedian-normalized for each cell line sample. Three sets of microarrayprofile comparisons were generated: (1) MeWo control cells relative toMeWo cells over-expressing miR-199a or miR-1908, (2) MeWo-LM2 controlcells relative to MeWo-LM2 cells expressing a short hairpin (miR-Zip)targeting miR-199a-3p, miR-199a-5p, or miR-1908, and (3) MeWo parentalcells relative to MeWo-LM2 cells. Based on the median-normalizedexpression values from these arrays, the following criteria were used toarrive at possible target genes common to miR-199a and miR-1908: (1)Genes down-regulated by more than 1.5 fold upon individualover-expression of each miR-199a and miR-1908, (2) Genes up-regulated bymore than 1.5 fold upon inhibition of either both miR-199a-3p andmiR-1908 or both miR-199a-5p and miR-1908, and (3) genes down-regulatedby more than 1.5 fold in LM2 cells, which express physiologically higherlevels of the three miRNAs, relative to MeWo parental cells.

Analysis of miRNA and mRNA Expression in Cell Lines

Total RNA was extracted from various cell lines using the miRvana kit(AM1560, Applied Biosystems, Austin, Tex.). The expression levels ofmature miRNAs were quantified using the Taqman miRNA expression assay(4427975-0002228, Applied Biosystems). RNU44 was used as an endogenouscontrol for normalization. For mRNA expression analyses, 600 ng of totalRNA was reverse transcribed using the cDNA First-Strand Synthesis Kit(18080-051, Invitrogen), and roughly 200 ng of the resulting cDNA wasthen mixed with SYBR green PCR Master Mix (4309155, Applied Biosystems)and the appropriate primers. Each reaction was performed inquadruplicate, and mRNA expression was quantified by performingreal-time PCR amplification using an ABI Prism 7900HT Real-Time PCRSystem (Applied Biosystems). GAPDH was used as an endogenous control fornormalization. The following primers were used:

ApoE_Fwd: 5′-TGGGTCGCTTTTGGGATTAC-3′ ApoE_Rev:5′-TTCAACTCCTTCATGGTCTCG-3′ DNAJA4_Fwd: 5′-CCAGCTTCTCTTCACCCATG-3′DNAJA4_Rev: 5′-GCCAATTTCTTCGTGACTCC-3′ GAPDH_Fwd:5′-AGCCACATCGCTCAGACAC-3′ GAPDH_Rev: 5′-GCCCAATACGACCAAATCC-3′ LRP1_Fwd:5′-TTTAACAGCACCGAGTACCAG-3′ LRP1_Rev: 5′CAGGCAGATGTCAGAGCAG-3′ LRP8_Fwd:5′-GCTACCCTGGCTACGAGATG-3′ LRP8_Rev: 5′-GATTAGGGATGGGCTCTTGC-3′

Elisa

Conditioned cancer cell media was prepared by incubating cells in 0.2%FBS serum starvation DMEM-based media for 24 hours. ApoE levels inconditioned media were determined using the APOE ELISA kit (IRAPKT031,Innovative Research, Novi, Mich.).

Luciferase Reporter Assays

Heterologous luciferase reporter assays were performed as previouslydescribed (Tavazoie et al., 2008). In brief, full-length 3′UTRs andCDS's of ApoE and DNAJA4 were cloned downstream of a renilla luciferasereporter into the psiCheck2 dual luciferase reporter vector (C8021,Promega, Madison, Wis.). 5×10⁴ parental MeWo cells, MeWo-LM2 cells, MeWocells over-expressing miR-199a, miR-1908, or a control hairpin, andMeWo-LM2 cells expressing a miR-Zip hairpin targeting miR-199a-3p,miR-199a-5p, miR-1908, or a control sequence were transfected with 100ng of the respective specific reporter constructs using TransiT-293transfection reagent. Twenty-four hours post-transfection, the cellswere lysed, and the ratio of renilla to firefly luciferase expressionwas determined using the dual luciferase assay (E1910, Promega).Putative miRNA binding sites in each target construct were identified byalignment to the complimentary miRNA seed sequences (miR-199a-3p:5′-CAGUAGUC-3′; miR-199a-5p: 5′-CCAGUGUU-3′; miR-1908: 5′-GGCGGGGA-3′).The miRNA complimentary sites on each target construct were mutatedusing the QuickChange Multi Site-Directed Mutagenesis Kit (200514,Agilent Technologies, Santa Clara, Calif.). Based on miRNA seed sequencecomplimentarity analysis, the CDS of ApoE was mutated at position 141(CTG to ACT), the 3′UTR of ApoE was mutated at positions 83 (GCC to ATA)and 98 (CTG to ACA), the CDS of DNAJA4 was mutated at positions 373 (CGCto TAT) and 917 (CTG to AGA), and the 3′UTR of DNAJA4 was mutated atpositions 576 (CTG to ACA), 1096 (CTG to TCT), 1396 (CGC to TGT), and1596 (CTG to TGT). The following primers were used to clone the 3′UTR'sand CDS's of ApoE and DNAJA4:

ApoE_CDS_Fwd: 5′-AGTACCTCGAGGGGATCCTTGAGTCCTACTC-3′ APOE_CDS_Rev:5′-TAATTGCGGCCGCTCAGACAGTGTCTGCACCCAG-3′ DNAJA4_CDS_Fwd:5′-TAATATCTCGAGATGTGGGAAAGCCTGACCC-3′ DNAJA4_CDS_Rev:5′-CAATTGCGGCCGCTCATGCCGTCTGGCACTGC-3′ APOE_3′UTR_Fwd:5′-TTAGCCTCGAGACGCCGAAGCCTGCAGCCA-3′ APOE_3′UTR_Rev:5′-TTACTGCGGCCGCTGCGTGAAACTTGGTGAATCTT-3′ DNAJA4_3′UTR_Fwd:5′-TAATATCTCGAGCGTGGTGCGGGGCAGCGT-3′ DNAJA4_3′UTR_Rev:5′-CAATTGCGGCCGCTTATCTCTCATACCAGCTCAAT-3′

The following primers were used to mutagenize the miRNA binding sites oneach target:

APOE_CDS_mut: 5′-GCCAGCGCTGGGAACTGGCAACTGGTCGCTTTTGGGATTACCT-3′APOE_3′UTR_mut1: 5′-CAGCGGGAGACCCTGTCCCCATACCAGCCGTCCTCCTGGGGTG-3′APOE_3′UTR_mut2: 5′-TCCCCGCCCCAGCCGTCCTCACAGGGTGGACCCTAGTTTAATA-3′DNAJA4_CDS_mut1: 5′-GGGATCGGTGGAGAAGTGCCTATTGTGCAAGGGGCGGGGGATG-3′DNAJA4_CDS_mut2: 5′-GTAGGGGGCGGGGAACGTGTTATCCGTGAAGAGGTGGCTAGGG-3′DNAJA4_3′UTR_mut1: 5′-CAGGGCCAACTTAGTTCCTAACATTCTGTGCCCTTCAGTGGAT-3′DNAJA4_3′UTR_mut2: 5′-ACAGTTTGTATGGACTACTATCTTAAATTATAGCTTGTTTGGA-3′DNAJA4_3′UTR_mut3: 5′-TAATTATTGCTAAAGAACTATGTTTTAGTTGGTAATGGTGTAA-3′DNAJA4_3′UTR_mut4: 5′-CAGCTGCACGGACCAGGTTCCATAAAAACATTGCCAGCTAGTGAG-3′Analysis of miRNA Expression in Human Melanoma Skin Lesions

All human clinical samples used in this study were obtained, processed,and analyzed in accordance with institutional IRB guidelines.Paraffin-embedded cross-sections of primary melanoma skin lesions from71 human patients were obtained from MSKCC. The samples werede-paraffinized by five consecutive xylene washes (5 minutes each).Following de-paraffinization, the malignancy-containing region wasidentified by H&E staining, dissected, and total RNA was extracted fromit using the RecoverAll Total Nucleic Acid Isolation Kit (AM1975,Applied Biosystems). The expression levels of mature miR-199a-3p,miR-199a-5p, and miR-1908 in each sample were quantified in a blindedfashion using the Taqman miRNA assay. RNU44 was used as an endogenouscontrol for normalization. The expression levels of each miRNA werecompared between primary melanomas with propensity to metastasize andprimary melanomas that did not metastasize. Kaplan-Meier curves wereplotted using metastasis-free survival data of patients as a function ofthe expression levels for each miRNA in each patient's tumor. Metastaticrecurrence to such sites as lung, brain, bone, and soft tissue werepreviously documented and allowed for a retrospective analysis of therelationship between the expression levels of identified miRNAs andmetastatic recurrence.

Histology

Animals were perfused with PBS followed by fixation with 4%paraformaldehyde infused via intracardiac and subsequently intratrachealinjection. The lungs were sectioned out, incubated in 4%paraformaldehyde at 4° C. overnight, embedded in paraffin, and slicedinto 5-μm-thick increments. For gross macroscopic metastatic nodulevisualization, lung sections were H&E stained. For endothelial contentanalysis in metastatic nodules formed by human melanoma MeWo cells inmice, representative lung sections were double-stained with primaryantibodies against MECA-32 (Developmental Studies Hybridoma Bank, TheUniversity of Iowa, IA), which labels mouse endothelial cells, and humanvimentin (VP-V684, Vector Laboratories), which labels human melanomacells. Various Alexa Flour dye-conjugated secondary antibodies were usedto detect primary antibodies. To determine the blood vessel densitywithin metastatic nodules, fluorescence was measured using a Zeiss laserscanning confocal microscope (LSM 510), and the MECA-32 signal withineach metastatic nodule, outlined based on co-staining with humanvimentin, was quantified in a blinded fashion using ImageJ (NIH). Forendothelial content analysis in metastatic nodules formed by mouseB16F10 mouse melanoma cells in wild type and ApoE genetically null mice,representative lung sections were stained for MECA-32, and the MECA-32signal within each nodule, demarcated based on cell pigmentation, wasquantified in a blinded fashion. The collective vessel area, given asthe percentage area covered by blood vessels relative to the total areaof each metastatic nodule, was obtained by background subtraction(rolling ball radius of 1 pixel) and use of a pre-determined thresholdas a cut-off. A metastatic nodule was defined as any region of greaterthan 2000 μm² total area. For large nodules, minimum of fourrepresentative images were obtained, and their average blood vesseldensity was calculated.

In Vivo Matrigel Plug Assay

10 μg/mL recombinant human ApoE3 (4696, BioVision), 10 μg/mL BSA (A2153,Sigma Aldrich), or 400 ng/ml VEGF were mixed with matrigel (356231, BDBiosciences) as indicated. 400 μL of matrigel containing the indicatedrecombinant proteins were injected subcutaneously just above the ventralflank of immunocompromised NOD-SCID mice. Plugs were extracted on day 3post-injection and fixed in 4% paraformaldehyde for 48 hours. Plugs werethen paraffin-embedded and sectioned at 5-μm-thick increments. Plugcross-sectional sections were immunohistochemically stained using aprimary antibody against the mouse endothelial antigen MECA-32(Developmental Studies Hybridoma Bank, The University of Iowa, IA),detected by peroxidase-conjugated secondary antibody, and subsequentlyvisualized by DAB oxidization. To quantify the extent of endothelialcell invasion into each matrigel plug, the number of endothelial cellswas counted in 4-5 random fields for each plug, and the average numberof endothelial cells per given plug area was calculated.

Tissue Culture

The SK-Mel-334 primary human melanoma line was established from a softtissue metastasis of a Braf-mutant melanoma of a patient at the MSKCC.Following minimum expansion in vitro, the cells were in vivo selected(Pollack and Fidler, 1982) to generate the lung-metastatic derivativesSK-Mel-334.2. The SK-Mel-239 vemurafenib-resistant clone (C1) was a giftfrom Poulikos Poulikakos (Mount Sinai Medical School) and theB-Raf^(V600E/+); Pten^(−/−); CDKN2A^(−/−) primary murine melanoma cellline was generously provided by Marcus Rosenberg (Yale University). Allother cell lines used were purchased from ATCC.

ApoE Elisa

Extracellular ApoE levels in serum-free conditioned media from melanomacells treated with DMSO, GW3965, or T0901317 (1 μM each) were quantifiedusing the ApoE ELISA kit (Innovative Research) at 72 hours followingtreatment.

Western Blotting

Mouse lung and brain tissue samples were homogenized on ice in RIPAbuffer (Sigma-Aldrich) supplemented with protease inhibitors (Roche).Mouse adipose tissue was homogenized on ice in TNET buffer (1.5 mM TrispH 7.5, 150 mM NaCl, 2 mM EDTA 1% triton, protease inhibitors). Totalprotein lysate (2 μg) was separated by SDS-PAGE, transferred to PVDFmembrane, and blotted with an anti-mouse ApoE (ab20874, Abcam) andanti-tubulin α/β (2148, Cell Signaling) antibodies.

ApoE Expression Analysis in Melanoma Clinical Samples

All clinical sample procurement, processing, and analyses were performedin strict agreement with IRB guidelines. Primary melanoma skin lesionswere previously resected from patients at the MSKCC, formalin-fixed,paraffin-embedded, and sectioned into 5-μm-thick slides. ApoE proteinexpression was assessed by double-blinded immunohistochemical analysisusing the D6E10 anti-ApoE antibody (ab1906, Abcam).

Histochemistry

Animals were intracardially perfused with PBS followed by 4%paraformaldehyde (PFA). Fixed lungs were embedded in paraffin andsectioned into 5-μm-thick increments. Macroscopic lung metastaticnodules were visualized by H&E staining. For analysis of tumorendothelial cell content, proliferation, and apoptosis, primary tumorparaffin-embedded sections were stained with antibodies against MECA-32(Developmental Studies Hybridoma Bank, University of Iowa), KI-67(ab15580, Abcam), and cleaved caspase-3 (9661, Cell Signaling),respectively.

Tail-Vein Metastasis Assays

Melanoma cells used for in vivo metastasis assays were transduced with astably expressed retroviral construct encoding a luciferase reportergene (Ponomarev et al., 2004), allowing us to monitor the in vivoprogression of melanoma cells by bioluminescence imaging. The followingnumbers of melanoma cells, resuspended in 100 μL of PBS, were injectedintravenously via the tail-vein: 4×10⁴ MeWo cells, 2.5×10⁵ HT-144 cells,2×10⁵ SK-Mel-334.2 cells, 5×10⁴B16F10 cells, and 1×10⁵YUMM cells. TheMeWo, HT-144, and SK-Mel-334.2 cells were injected into 6-8 week-oldsex-matched NOD scid mice, while the B16F10 and YUMM cells were injectedinto 6-8 week-old sex-matched C57BL/6 mice. In all experiments assessingat the effects of GW3965 on metastasis formation, mice were pre-treatedon a control diet or a GW3965-supplemented diet (20 mg/kg) for 10 days.To assess the effect of GW3965 treatment on brain metastasis, 1×10⁵ MeWobrain-metastatic derivatives were injected intracardially into athymicnude mice. Immediately following injection, mice were randomly assignedto a control diet or GW3965-supplemented diet (100 mg/kg). To determinewhether oral delivery of GW3965 can inhibit the progression of incipientmetastasis, NOD Scid mice were intravenously injected with 4×10⁴MeWocells and the cells were allowed to colonize the lungs for 42 days,after which mice were blindedly assigned to a control diet or aGW3965-supplemented diet (100 mg/kg) treatment.

Orthotopic Metastasis Assays

To determine the effect of GW3965 treatment on lung colonization bymelanoma cells dissociated from an orthotopic site, 1×10⁶ MeWo cellsexpressing a luciferase reporter were subcutaneously injected into bothlower flanks of NOD Scid mice. Upon the formation of tumors measuring˜300 mm³ in volume, the tumors were excised and the mice were randomlyassigned to a control diet or a GW3965-supplemented diet (100 mg/kg)treatment. One month after tumor excision, the lungs were extracted andlung colonization was measured by ex vivo bioluminescence imaging. Tohistologically confirm the extent of melanoma lung colonization, lungswere then fixed in 4% PFA overnight, paraffin-embedded, section into5-μM increments and stained for human vimentin (VP-V684, VectorLaboratories).

Generation of Dacarbazine-Resistant Melanoma Cells

Dacarbazine-resistant B16F10 mouse melanoma cells were generated bycontinuously culturing the cells in the presence of DTIC (D2390,Sigma-Aldrich, St. Louis, Mo.). First, the cells were treated with 500μg/mL DTIC for one week. Following this initial DTIC treatment, theremaining (˜10%) viable cells were allowed to recover for one week,after which 750 μg/mL of DTIC was added to the cell media for 5 days.Subsequent to this high-dose treatment, the cells were allowed torecover in the presence of low-dose DTIC (100 μg/mL) for one week. Thecells were then continuously cultured in cell media containing 200 μg/mLDTIC for at least one month prior to grafting the cells into mice. DTICwas added to fresh cancer cell media every 3 days. For tumor growthexperiments, 5×10⁴ B16F10 parental and DTIC-resistant cells weresubcutaneously injected into the lower flank of 7-week-old C57BL/6 mice.Following formation of small tumors measuring 5-10 mm³ in volume, themice were randomly assigned to the following treatment groups: (1)control diet+vehicle, i.p.; (2) control diet+DTIC i.p. (50 mg/kg); (3)GW3965-supplemented diet (100 mg/kg)+vehicle i.p. DTIC was dissolved inthe presence of citric acid (1:1 by weight) in water and administereddaily by intraperitoneal injection.

The DTIC-resistant MeWo human melanoma cell line clone was generatedfollowing DTIC treatment of mice bearing MeWo tumors measuring 600-800mm³ in volume. After initial tumor shrinkage in response to daily DTICdosing (50 mg/kg, i.p.) during the first two weeks, the tumorseventually developed resistance and resumed growth, at which point tumorcells were dissociated and the DTIC-resistant MeWo cell line wasestablished. The cells were expanded in vitro in the presence of DTIC(200 μg/mL) for one week, after which 5×10⁵ DTIC-resistant MeWo cellswere re-injected into 8-week old Nod SCID gamma mice. Following growthof tumors to 5-10 mm³ in volume, mice were blindedly assigned to thefollowing treatment groups: (1) control diet; (2) control diet+DTIC (50mg/kg); (3) GW3965-supplemented diet (100 mg/kg). To determine theeffect of DTIC on tumor growth by parental unselected MeWo cells, 5 10⁵MeWo cells were subcutaneously injected into Nod SCID gamma mice, andthe mice were treated with a control vehicle or DTIC (50 mg/kg)subsequent to formation of tumors measuring 5-10 mm³ in volume. DTIC wasadministered daily, as described above, in cycles consisting of 5consecutive daily treatments interspersed by 2-day off-treatmentintervals. Tumor growth was measured twice a week.

Genetically-Initiated Model of Melanoma Progression

The Tyr::CreER; B-Raf^(V600E/+); Pten^(lox/+)/Tyr::CreER;B-Raf^(V600E/+); Pten^(lox/lox) conditional model of melanomaprogression was previously established and characterized by Dankort etal. (2009). Briefly, melanoma in these mice was induced at 6 weeks ofage by intraperitoneally injecting 4-HT (H6278, 70% isomer,Sigma-Aldrich, St Louis, Mo.) at 25 mg/kg administered in peanut oil onthree consecutive days. The 4-HT stock solution was prepared bydissolving it in 100% EtOH at 50 mg/mL by heating at 45° C. for 5 minand mixing. Once dissolved, the stock 4-HT solution was then diluted by10-fold in peanut oil, yielding a 5 mg/mL 4-HT working solution that wasthen injected into mice. After the first 4-HT injection, mice wereblindedly assigned to receive either a control diet or a dietsupplemented with GW3965 (100 mg/kg). Mice were examined three times aweek for the presence and progression of melanoma lesions. At day 35,dorsal skin samples were harvested from control-treated andGW3965-treated mice, fixed in 4% PFA and photographed at 10×. Thepercentage of pigmented melanoma lesion area out of the total skin areawas quantified using ImageJ. For survival analyses, mice were monitoreddaily for melanoma progression and euthanized according to a standardbody condition score, taking into account initial signs of moribundstate and discomfort associated with the progression of melanoma burden.Post-mortem, the lungs, brains, and salivary glands were harvested andexamined for the presence of macroscopic melanoma lesions.

Mouse Genotyping

All mouse genotyping was performed using standard PCR conditions, asrecommended by Jackson Labs. The following genotyping primers were usedfor the respective PCR reactions: Tyr::CreER; B-Raf^(V600E/+);Pten^(lox/+) and Tyr::CreER; B-Raf^(V600E/+); Pten^(lox/lox) mice:

B-Raf Forward: 5′-TGA GTA TTT TTG TGG CAA CTG C-3′ B-Raf Reverse:5′-CTC TGC TGG GAA AGC GGC-3′ Pten Forward:5′-CAA GCA CTC TGC GAA CTG AG-3′ Pten Reverse:5′-AAG TTT TTG AAG GCA AGA TGC-3′ Cre Transgene Forward:5′-GCG GTC TGG CAG TAA AAA CTA TC-3′ Cre Transgene Reverse:5′-GTG AAA CAG CAT TGC TGT CAC TT-3′ Internal Positive Control Forward:5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′ Internal Positive Control Reverse:5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′ApoE−/− mice:

Common Forward: 5′-GCC TAG CCG AGG GAG AGC CG-3′ Wild-type Reverse:5′-TGT GAC TTG GGA GCT CTG CAG C-3′ Mutant Reverse:5′-GCC GCC CCG ACT GCA TCT-3′LXRα−/− mice:

Common Forward: 5′-TCA GTG GAG GGA AGG AAA TG-3′ Wild-type Reverse:5′-TTC CTG CCC TGG ACA CTT AC-3′ Mutant Reverse:5′-TTG TGC CCA GTC ATA GCC GAA T-3′LXRβ−/− mice:

Common Forward: 5′-CCT TTT CTC CCT GAC ACC G-3′ Wild-type Reverse:5′-GCA TCC ATC TGG CAG GTT C-3′ Mutant Reverse:5′-AGG TGA GAT GAC AGG AGA TC-3′

Cell Proliferation and Viability Assay:

To determine the effects of GW3965, T0901317, and Bexarotene on in vitrocell growth, 2.5×10⁴melanoma cells were seeded in triplicate in 6-wellplates and cultured in the presence of DMSO, GW3965, T0901317, orBexarotene at 1 μM each. After 5 days, the number of viable and deadcells was counted using the trypan blue dye (72-57-1, Sigma-Aldrich),which selectively labels dead cells.

Cell Invasion Assay

The cell invasion assay was performed as previously described in detail(Pencheva et al., 2012) using a trans-well matrigel invasion chambersystem (354480, BD Biosciences). In brief, various melanoma cells werecultured in the presence of DMSO, GW3965, T0901317, or Bexarotene at 1μM for 56 hours, after which melanoma cells were switched to starvationmedia (0.2% FBS) for 16 hours in the presence of each drug. Followingstarvation, cells were seeded into matrigel-coated trans-well inserts,and the invasion assay was allowed to proceed for 24 hours at 37° C. ForApoE antibody neutralization experiments, 40 μg/mL 1D7 anti-ApoEblocking antibody (Heart Institute, University of Ottawa, Ottawa,Canada) or 40 μg/mL anti-IgG control antibody (AB-108-C, R&D Systems,Minneapolis, Minn.) was added to each trans-well insert at the start ofthe assay.

Endothelial Recruitment Assay

The endothelial recruitment assay was carried out as previouslydescribed (Pencheva et al., 2012; Png et al., 2012). Melanoma cells weretreated with DMSO, GW3965, T0901317, or Bexarotene at 1 μM for 56 hours,after which 5×10⁴ cells were seeded in a 24-well plate in the presenceof each drug and allowed to attach for 16 hours prior to starting theassay. HUVEC cells were serum-starved overnight in EGM-2 mediacontaining 0.2% FBS. The following day, 1×10⁵ HUVEC cells were seededinto a 3.0 μm HTS Fluoroblock trans-well migration insert (351151, BDFalcon, San Jose, Calif.) fitted into each well containing cancer cellsat the bottom. The HUVEC cells were allowed to migrate towards thecancer cells for 20 hours at 37° C., after which the inserts wereprocessed as previously described (Pencheva et al., 2012). For ApoEantibody neutralization experiments, 40 μg/mL 1D7 anti-ApoE blockingantibody (Heart Institute, University of Ottawa, Ottawa, Canada) or 40μg/mL anti-IgG control antibody (AB-108-C, R&D Systems, Minneapolis,Minn.) was added to each trans-well insert at the start of the assay.

Lentiviral shRNA-Based Gene Knockdown

shRNAs were integrated into lentiviral particles that were prepared bytransfection of 6 of vector A, 12 μg of vector K, and 12 μg of shRNAplasmid into HEK-293T packaging cells, as previously described (Penchevaet al., 2012; Png et al., 2012). Lentiviral shRNA transduction wasperformed in the presence of 10 μg/mL of polybrene (TR-1003-G,Millipore, Billerica, Mass.) for 6 hours, as described previously(Pencheva et al., 2012). The cells were expanded for 72 hours aftertransduction and lentiviral selection was performed by culturing thecells in the presence of 2 μg/mL of puromycin (P8833, Sigma-Aldrich) for72 hours.

The following shRNA sequences were used:

Human:

sh₁LXRα: 5′-CCGGCCGACTGATGTTCCCACGGATCTCGAGATCCGTGGGAACATCAGTCGGTTTTT-3′ sh₂LXRα:5′-CCGGGCAACTCAATGATGCCGAGTTCTCGAGAACTCGGCATCA TTGAGTTGCTTTTT-3′sh₁LXRβ: 5′-CCGGAGAGTGTATCACCTTCTTGAACTCGAGTTCAAGAAGGTGATACACTCTTTTTT-3′ sh₂LXRβ:5′-CCGGGAAGGCATCCACTATCGAGATCTCGAGATCTCGATAGTG GATGCCTTCTTTTT-3′ shApoE:5′-CCGGGCAGACACTGTCTGAGCAGGTCTCGAGACCTGCTCAGAC AGTGTCTGCTTTTT-3′

Mouse:

sh_mLXRα: 5′-CCGGGCAACTCAATGATGCTGAGTTCTCGAGAACTCAGCATCATTGAGTTGCTTTTT-3′ sh_mLXRβ:5′-CCGGTGAGATCATGTTGCTAGAAACCTCGAGGTTTCTAGCAAC ATGATCTCATTTTTG-3′sh_mApoE: 5′-CCGGGAGGACACTATGACGGAAGTACTCGAGTACTTCCGTCATAGTGTCCTCTTTTT-3′Gene Expression Analysis by qRT-PCR:

RNA was extracted from whole cell lysates using the Total RNAPurification Kit (17200, Norgen, Thorold, Canada). 600 ng of total RNAwas then reverse transcribed into cDNA using the cDNA First-StrandSynthesis Kit (18080-051, Invitrogen), and quantitative real-time PCRamplification was performed as previously described (Pencheva et al.,2012) using an ABI Prism 7900HT Real-Time PCR System (AppliedBiosystems, Austin, Tex.). Each PCR reaction was carried out inquadruplicates. Gene expression was normalized to GAPDH, which was usedas an endogenous control.

The following primers were used:

Human:

ApoE Forward: 5′-TGGGTCGCTTTTGGGATTAC-3′ ApoE Reverse:5′-TTCAACTCCTTCATGGTCTCG-3′ GAPDH Forward: 5′-AGCCACATCGCTCAGACAC-3′GAPDH Reverse: 5′-GCCCAATACGACCAAATCC-3′ LXRα_Fwd:5′-GTTATAACCGGGAAGACTTTGC-3′ LXRα_Rev: 5′-AAACTCGGCATCATTGAGTTG-3′LXRβ_Fwd: 5′-TTTGAGGGTATTTGAGTAGCGG-3′ LXRβ_Rev:5′-CTCTCGCGGAGTGAACTAC-3′

Mouse:

ApoE Forward: 5′-GACCCTGGAGGCTAAGGACT-3′ ApoE Reverse:5′-AGAGCCTTCATCTTCGCAAT-3′ GAPDH Forward: 5′-GCACAGTCAAGGCCGAGAAT-3′GAPDH Reverse: 5′-GCCTTCTCCATGGTGGTGAA-3′ LXRα Forward:5′-GCGCTCAGCTCTTGTCACT-3′ LXRα Reverse: 5′-CTCCAGCCACAAGGACATCT-3′ LXRβForward: 5′-GCTCTGCCTACATCGTGGTC-3′ LXRβ Reverse:5′-CTCATGGCCCAGCATCTT-3′ ABCA1 Forward: 5′-ATGGAGCAGGGAAGACCAC-3′ABCA1 Reverse: 5′-GTAGGCCGTGCCAGAAGTT-3′

ApoE Promoter Activity Assay

The ApoE promoter, consisting of a sequence spanning 980 base pairsupstream and 93 base pairs downstream of the ApoE gene, was cloned intoa pGL3-Basic vector (E1751, Promega Corporation, Madison, Wis.) upstreamof the firefly luciferase gene using NheI and SacI restriction enzymes.Then, multi-enhancer elements 1 (ME.1) and 2 (ME.2) were cloned directlyupstream of the ApoE promoter using MluI and SacI restriction enzymes.To assess ApoE promoter- and ME.1/ME.2-driven transcriptional activationby LXR agonists, 5×10⁴ MeWo cells were seeded into a 24-well plate. Thefollowing day, 100 ng of pGL3-ME.1/ME.2-ApoE promoter construct and 2 ngof pRL-CMV renilla luciferase construct (E2261, Promega) wereco-transfected into cells in the presence of DMSO, GW3965, or T0901317at 1 μM, each condition in quadruplicate. To assess transcriptionalactivation by LXRα or LXRβ, 5×10⁴ MeWo cells expressing a control shRNAor shRNA targeting LXRα or LXRβ were seeded into a 24-well plate. Thefollowing day, 200 ng of pGL3-ME.1/ME.2-ApoE promoter construct and 2 ngof pRL-CMV renilla luciferase were co-transfected into cells in thepresence of DMSO, GW3965, or T0901317 at 1 μM, each condition inquadruplicate. After 24 hours, cells were lysed, and cell lysate wasanalyzed for firefly and renilla luciferase activity using the DualLuciferase Assay System (E1960, Promega) and a Bio-Tek Synergy NEOMicroplate Reader. Firefly luciferase signal was normalized to renillaluciferase signal and all data are expressed relative to the luciferaseactivity ratio measured in the DMSO-treated control cells.

The following cloning primers were used:

ApoE-promoter Forward: 5′-TCA TAG CTA GCG CAG AGC CAG GAT TCA CGCCCT G-3′ ApoE-promoter Reverse:5′-TGG TCC TCG AGG AAC CTT CAT CTT CCT GCC TGT GA-3′ ME.1 Forward:5′-TAG TTA CGC GTA GTA GCC CCC ATC TTT GCC-3′ ME.1 Reverse:5′-AAT CAG CTA GCC CCT CAG CTG CAA AGC TC-3′ ME.2 Forward:5′-TAG TTA CGC GTA GTA GCC CCC TCT TTG CC-3′ ME.2 Reverse:5′-AAT CAG CTA GCC CTT CAG CTG CAA AGC TCT G-3′

Tumor Histochemistry

Tumors were excised from mice and fixed in 4% paraformaldehyde at 4° C.for 48 hours. Then, tumors were paraffin-embedded and sectioned into5-μm-thick increments. For endothelial cell content analysis in tumors,tumor sections were stained with a primary antibody against the mouseendothelial cell marker MECA-32 (Developmental Studies Hybridoma Bank,The University of Iowa, IA) and counterstained with DAPI nuclear stain.To determine tumor cell proliferation and apoptosis, tumor sections werestained with antibodies against the proliferative marker Ki-67 (Abcam,ab15580, Cambridge, Mass.) and the apoptotic marker cleaved caspase-3(9661, Cell Signaling, Danvers, Mass.), respectively. Various AlexaFlour dye-conjugated secondary antibodies were used to detect primaryantibodies. Fluorescence was measured using inverted fluorescencemicroscope (Zeiss Axiovert 40 CFL) at 5× magnification for MECA-32 andKi-67 staining and 10× magnification for cleaved caspase-3 staining.Endothelial cell content density and tumor proliferation rate werequantified by calculating the average percentage of MECA-32 or Ki-67positively-staining area out of the total tumor area. Tumor apoptosiswas measured by counting the number of cleaved caspase-3 expressingcells per given tumor area.

Analysis of ApoE Expression in Primary Melanoma Lesions

Human primary melanoma skin samples were resected from melanoma patientsat MSKCC, formalin-fixed, embedded in paraffin, and sectioned into5-μm-thick increments. To determine ApoE protein expression, the sampleswere first de-paraffinized by two consecutive xylene washes (5 minuteseach), and rehydrated in a series of ethanol washes (100%, 95%, 80%, and70% EtOH). ApoE antigen was retrieved by incubating the samples in thepresence of proteinase K (5 μg/mL) for 20 minutes at room temperature.To quench endogenous peroxidase activity, the slides were incubated in3% H₂O₂ solution. The slides were then blocked in three consecutiveAvidin, Biotin, and horse serum block solutions for 15 min each at roomtemperature (SP-2001, Vector Laboratories, Burlingame, Calif.). ApoE wasdetected by staining with D6E10 anti-ApoE antibody (ab1908, Abcam),which was used at a 1:100 dilution in PBS at 4° C. overnight. Theprimary antibody was then recognized by incubating the slides in aperoxidase-conjugated secondary antibody (PK-4002, Vector Laboratories)and exposed by DAB (SK-4105, Vector Laboratories) oxidation reaction.The slides were imaged at 10× magnification and analysed in adouble-blinded manner. ApoE expression was quantified by counting thenumber of DAB-positive cells and measuring the area of extracellularApoE staining. Total ApoE staining signal was expressed as thepercentage staining area per given tumor area, determined based onmatched H&E-stained slides for each sample. Kaplan-Meier curvesdepicting patients' metastasis-free survival times were generated byplotting each patient's relapse-free survival data as a function of ApoEexpression in that patient's primary melanoma lesion. Patients whosetumors had ApoE levels lower than the median ApoE expression of thepopulation were classified as ApoE-negative, whereas patients whosemelanomas expressed ApoE above the median were classified asApoE-positive. Previously documented patients' history of metastaticrecurrence to sites such as lung, brain, bone, soft and subcutaneoustissues, and skin enabled us to retrospectively determine therelationship between ApoE expression at a primary melanoma site andmetastatic relapse.

Example 2 Endogenous Mir-1908, Mir-199a-3p, And Mir-199a-5p PromoteHuman Melanoma Metastasis

In order to identify miRNA regulators of melanoma metastasis, in vivoselection (Pollack and Fidler, 1982) was utilized with the pigmentedMeWo and non-pigmented A375 human melanoma cell lines to generatemultiple second (LM2) and third generation (LM3) lung metastaticderivatives. Comparison of the metastatic potential of the MeWo-LM2 andA375-LM3 lines showed these derivatives to metastasize significantlymore efficiently than their respective parental populations in lungcolonization assays (FIGS. 12A-B). Hybridization-based small RNAprofiling of 894 mature miRNAs followed by quantitative stem-loop PCR(qRT-PCR) revealed four miRNAs (miR-1908, miR-199a-3p, miR-199a-5p, andmiR-214) to be upregulated greater than two-fold in multiple A375 andMeWo metastatic derivatives relative to their respective parental cells(FIGS. 1A-B, 12C). The significant induction of miR-199a-3p,miR-199a-5p, miR-214, and miR-1908 across multiple metastaticderivatives suggested a metastasis-promoting role for these miRNAs.Retrovirally mediated transduction and over-expression of the precursorsfor miR-199a-3p and miR-199a-5p (over-expressed concomitantly as themiR-199a hairpin) and miR-1908 lead to a robust increase in lungmetastatic colonization based on both bioluminescence signalquantification and gross lung histology (FIG. 1C, 12D; 9.64-foldincrease, P=0.016 for miR-1908; 8.62-fold increase, P=0.028 formiR-199a), while miR-214 over-expression did not significantly affectmetastasis. Importantly, over-expression of each miR-199a and miR-1908increased the number of metastatic nodules formed (FIG. 12E), consistentwith a role for these miRNAs in metastatic initiation. These findingsalso revealed miR-199a and miR-1908 to be sufficient for enhancedmetastatic colonization.

Next, assays were carried out to examine if endogenous levels of thesemiRNAs promote metastasis. To this end, miR-1908 and each of the twomiRNAs arising from the miR-199a hairpin (miR-199a-3p and miR-199a-5p)were inhibited in the highly metastatic cells through miR-Ziptechnology. Individual inhibition of each of these miRNAs suppressedmetastatic colonization by more than 7-fold (FIG. 1D; P=0.047 formiR-1908 inhibition; P=0.010 for miR-199a-3p inhibition; P=0.015 formiR-199a-5p inhibition) and dramatically decreased the number ofmetastatic nodules formed (FIG. 12F).

To determine whether these miRNAs also promote metastasis in anindependent cell line, their expression was silenced in the A375metastatic derivative cell line. Indeed, inhibition of miR-1908,miR-199a-3p, or miR-199a-5p significantly reduced the lung colonizationcapacity of metastatic A375-LM3 cells (FIG. 1E), establishing thesethree miRNAs as endogenous promoters of metastasis by human melanomacells.

Given the robust functional roles of miR-1908, miR-199a-3p, andmiR-199a-5p in promoting melanoma metastasis in a mouse model of humancell metastasis, further assays were carried out to examine whetherexpression of these miRNAs correlates with the capacity of human primarymelanoma lesions to metastasize. To this end, 71 primary melanoma skinlesions obtained from Memorial Sloan-Kettering Cancer Center (MSKCC)patients were analyzed in a blinded fashion for the expression levels ofmiR-1908, miR-199a-3p, and miR-199a-5p through qRT-PCR. Consistent withthe above functional studies, all three miRNAs were significantlyinduced in primary melanomas that had metastasized relative to thosethat had not (FIG. 1F; P=0.037 for miR-1908; P=0.0025 for miR-199a-3p;P=0.0068 for miR-199a-5p), suggesting that upregulated expression ofthese miRNAs in primary lesions is an early event predictive of melanomacancer progression.

Example 3 Mir-1908, Mir-199a-3p, and Mir-199a-5p Promote Cell Invasionand Endothelial Recruitment

In this Examiner, assays were carried out to determine the cellularmechanisms by which miR-1908, miR-199a-3p, and miR-199a-5p regulatemetastasis.

First, it was examined if these miRNAs promote metastasis by enhancingproliferation or tumor growth. Contrary to this, over-expression of eachmiRNA reduced cell proliferation (FIG. 13A). More importantly, miR-1908over-expression did not increase primary tumor growth, while miR-199aover-expression actually lead to a significant decrease (35%; P<0.001)in tumor volume (FIG. 2A), indicating that the pro-metastatic effects ofmiR-1908 and miR-199a are not secondary to tumor growth promotion orenhanced cell proliferation.

Next, it was examined whether these miRNAs regulate cell invasion, a keymetastatic phenotype. Metastatic LM2 cells, which express higher levelsof these miRNAs, displayed significantly increased matrigel invasioncapacity relative to their less metastatic parental population (FIG.13B). Accordingly, over-expression of miR-199a and miR-1908 individuallyenhanced the ability of parental MeWo cells to invade through matrigel(FIG. 2B; three-fold increase for miR-199; two-fold increase formiR-1908). Conversely, individual inhibition of miR-199a-3p,miR-199a-5p, and miR-1908 significantly decreased the invasive capacityof MeWo-LM2 (FIG. 2C) as well as A375-LM3 (FIG. 2D) metastatic melanomacell derivatives.

Given the robust effects of these miRNAs on metastatic progression,further analyses were conducted to examiner whether they may regulateany additional pro-metastatic phenotypes. While over-expression ofmiR-199a or miR-1908 did not modulate melanoma cell adhesion toendothelial cells (FIG. 13C), resistance to anoikis (FIG. 13D), survivalin the setting of serum starvation (FIG. 13E), or colony formation (FIG.13F), each miRNA dramatically enhanced (more than three-fold increase)the ability of parental MeWo cells to recruit endothelial cells intrans-well endothelial recruitment assays (FIG. 2E). Consistent withthis, metastatic Mewo-LM2 cells, which physiologically over-expressmiR-199a and miR-1908, were more efficient at recruiting endothelialcells relative to their parental cells (FIG. 13G). Conversely,inhibition of miR-199a-3p, miR-199a-5p, or miR-1908 in the metastaticMeWo-LM2 (FIG. 2F) as well as A375-LM3 cells (FIG. 2G) suppressedendothelial recruitment, consistent with the requirement and sufficiencyof these miRNAs for enhanced endothelial recruitment capacity ofmetastatic melanoma cells.

To determine whether endogenous miR-199a-3p, miR-199a-5p, and miR-1908regulate endothelial recruitment by metastatic cells in vivo, assayswere carried out to examine metastatic blood vessel density byperforming co-immunostaining for human vimentin, which labels human MeWomelanoma cells, and mouse endothelial cell antigen (MECA-32), whichlabels mouse endothelial cells. Strikingly, inhibition of miR-199a-3p,miR-199a-5p, or miR-1908 individually led to pronounced decreases (anaverage of 3-fold for miR-199a-3p and miR-199a-5p and 4.7-fold formiR-1908) in blood vessel density within metastatic nodules (FIG. 2H;P<0.001 for miR-199a-3p; P<0.001 for miR-199a-5p; and P<0.001 formiR-1908), revealing a role for these miRNAs in promoting metastaticendothelial content and metastatic angiogenesis. Conversely,over-expression of each miRNA in poorly metastatic melanoma cellsdramatically increased metastatic blood vessel density (FIG. 13H). Thesefindings reveal miR-199a-3p, miR-199a-5p, and miR-1908 as necessary andsufficient for enhanced invasion and endothelial recruitment duringmelanoma progression.

Example 4 Mir-1908, Mir-199a-3p, and Mir-199a-5p Convergently andCooperatively Target Apoe and DNAJA4

In this example, a systematic and unbiased approach was employed toidentify the direct molecular targets of these miRNAs.

Since miR-1908, miR-199a-3p, and miR-199a-5p mediate the same sets of invitro and in vivo phenotypes and miR-199a-5p and miR-199a-3p arise fromthe same precursor hairpin, it was hypothesized that the pro-metastaticphenotypes of these miRNAs may arise through silencing of common targetgenes. Given that mammalian miRNAs act predominantly by destabilizingtarget mRNA transcripts (Guo et al., 2010 Nature 466, 835-840),transcriptomic profiling of melanoma cells was performed in the contextof both loss- and gain-of-function for each miRNA. This revealed a smallset of genes that were repressed by both miR-199a and miR-1908 and thatalso displayed lower levels in the metastatic LM2 derivatives, whichexpress higher endogenous levels of these miRNAs (FIG. 14A).Quantitative RT-PCR validated two genes, the metabolic geneApolipoprotein E (ApoE) and the heat-shock protein DNAJA4, assignificantly modulated by miR-199a and miR-1908 and dramaticallysilenced in the highly metastatic LM2 cells (FIGS. 3A and 14B-D).

To determine whether ApoE and DNAJA4 are directly targeted by miR-1908,miR-199a-3p, and miR-199a-5p, the effects of each miRNA on the stabilityof its putative targets were examined through heterologous luciferasereporter assays. Interestingly, over-expression of miR-199a repressedthe stability of the 3′ untranslated region (UTR) and coding sequence(CDS) of both ApoE and DNAJA4, while over-expression of miR-1908destabilized the 3′UTR of ApoE and the 3′UTR and CDS of DNAJA4.Consistent with direct targeting, mutating the miRNA complementarysequences on each target abrogated miRNA-mediated regulation (FIG. 3B).In a direct test of endogenous targeting, individual miRNA inhibition inmetastatic LM2 cells resulted in increased target stability (FIG. 3C)that was abrogated upon mutating the miRNA target sites (FIG. 14E),revealing ApoE to be directly targeted by miR-1908 and miR-199a-5p andDNAJA4 to be directly targeted by all three miRNAs (FIG. 3D).Importantly, the CDS's and 3′UTR's of both of these genes were lessstable in the highly metastatic LM2 cells, which express physiologicallyhigher levels of the three regulatory miRNAs, indicating that endogenoustargeting of ApoE and DNAJA4 by these miRNAs is relevant to melanomametastasis (FIG. 3E).

Given the molecular convergence of miR-199a-3p, miR-199a-5p, andmiR-1908 onto common target genes, it was next examined whether thesetargets, ApoE and DNAJA4, could mediate the metastatic phenotypesconferred by these miRNAs. Over-expression of each gene in themetastatic LM2 cells led to pronounced reductions in cell invasion andendothelial recruitment phenotypes (FIGS. 3F-G, 14F). Conversely,knock-down of ApoE or DNAJA4 in the poorly metastatic cells usingindependent hairpins significantly enhanced cell invasion andendothelial recruitment (FIGS. 3H-I, 14G), revealing ApoE and DNAJA4 toact as endogenous suppressors of these pro-metastaticphenotypes—consistent with their targeting by the above mentionedmetastasis-promoting miRNAs.

Example 5 ApoE and DNAJA4 Mediate miR-199a- and miR-1908-DependentMetastatic Invasion, Endothelial Recruitment, and Colonization

To determine whether ApoE and DNAJA4 are the direct biological effectorsdownstream of miR-199a and miR-1908, assays were carried out to examinewhether these two target genes epistatically interact with each miRNA.As expected, miRNA silencing reduced the invasion and endothelialrecruitment capacity of highly metastatic melanoma cells. Importantly,knock-down of ApoE or DNAJA4 in the setting of miRNA inhibitionsignificantly occluded the suppression of invasion (FIGS. 4A and 4C) andendothelial recruitment (FIGS. 4B and 4D) upon silencing of each miRNA.Strikingly, knock-down of either of these genes in cells depleted formiR-1908 or miR-199a-5p fully rescued the dramatic suppression ofmetastatic colonization resulting from miRNA inhibition (FIG. 4E-F,15E). Conversely, over-expression of ApoE or DNAJA4 in cellsover-expressing miR-1908 (FIG. 4G-H, 15F) or miR-199a (FIG. 15G-I) wassufficient to suppress cell invasion and endothelial recruitment.Additionally, ApoE or DNAJA4 over-expression was sufficient to inhibitmiRNA-mediated metastatic colonization (FIG. 15J). Importantly, ApoE andDNAJA4 were also required for miRNA-dependent enhanced cell invasion andendothelial recruitment by the highly metastatic A375-LM3 cells (FIGS. 4I-J, 15K).

To determine whether ApoE and DNAJA4 also regulate miRNA-dependentmetastatic endothelial recruitment in vivo, co-immunostaining ofmelanoma metastases (human vimentin) and endothelial cells (MECA-32) wasperformed in lung metastatic nodules formed by cells knocked-down foreach of these genes in the context of miRNA inhibition. Notably,knock-down of ApoE or DNAJA4 resulted in a significant (>3.5-fold)increase in metastatic blood vessel density in metastases arising fromcells with miRNA silencing (FIG. 4K, P<0.01 for both ApoE and DNAJA4knock-down cells). These findings reveal ApoE and DNAJA4 as directdownstream effectors of miRNA-dependent metastatic invasion,colonization, and endothelial recruitment phenotypes induced by thesepro-metastatic miRNAs in melanoma.

Example 6 Melanoma Cell-Secreted Apoe Is Both a Necessary and SufficientMediator of Invasion and Endothelial Recruitment, While Genetic Deletionof Apoe Promotes Metastasis

ApoE is a secreted factor. As such, it was examined whethermelanoma-cell secreted ApoE could suppress invasion and endothelialrecruitment. Accordingly, extracellular ApoE levels, detected by ELISA,were 3.5-fold lower in metastatic LM2 cells—which express higher levelsof miR-199a and miR-1908—than their less metastatic parental cells (FIG.5A). Secreted ApoE levels were also significantly suppressed byendogenous miR-199a and miR-1908 (FIGS. 5B and 16A).

Next, inhibiting ApoE through use of a neutralizing antibody (1D7) thatrecognizes the receptor-binding domain of ApoE enhanced both cellinvasion (FIG. 5C; 1.68-fold increase) and endothelial recruitment (FIG.5D; 1.84-fold increase) by parental MeWo cells, which express highendogenous levels of ApoE (FIG. 14C). Conversely, addition ofrecombinant human ApoE significantly suppressed invasion and endothelialrecruitment by metastatic LM2 cells (FIG. 5E), which exhibit lowendogenous ApoE levels (FIG. 14C). Importantly, recombinant ApoEaddition did not affect melanoma cell or endothelial cell in vitroproliferation (FIG. 16B-C) or survival in serum starvation conditions(FIG. 16D-E), indicating that suppression of these phenotypes byrecombinant ApoE is not secondary to a decrease in proliferation orimpaired survival. Consistent with ApoE being epistatically downstreamof miR-199a and miR-1908, neutralization of ApoE with the ApoEneutralizing antibody 1D7 significantly abrogated the suppressedinvasion and endothelial recruitment phenotypes seen with inhibition ofeach miRNA (FIGS. 5F-G). The above findings reveal melanomacell-secreted ApoE as a necessary and sufficient suppressor ofmiRNA-dependent invasion and endothelial recruitment phenotypes inmelanoma.

Further assays were carried out to investigate the mechanism by whichDNAJA4, a poorly characterized heat-shock protein, mediates endothelialrecruitment and invasion. Given the phenotypic commonalities displayedby ApoE and DNAJA4, it was hypothesized that DNAJA4 may play aregulatory role and enhance ApoE levels. Indeed, knock-down of DNAJA4reduced both ApoE transcript levels (FIG. 16F) as well as secreted ApoElevels (FIG. 5H), while DNAJA4 over-expression substantially elevatedApoE expression (FIG. 16G). Consistent with DNAJA4 acting upstream ofApoE, addition of recombinant ApoE abrogated the enhanced cell invasionand endothelial recruitment phenotypes seen with DNAJA4 knock-down (FIG.5I-J). Conversely, the suppression of invasion and endothelialrecruitment seen with DNAJA4 over-expression phenotypes weresignificantly occluded by antibody neutralization of ApoE (FIGS. 16H-I).These findings reveal DNAJA4 to suppress melanoma invasion andendothelial recruitment through positive regulation of ApoE expressionand resulting secretion.

In view of the regulatory convergence of three metastasis-promotingmiRNAs and the DNAJA4 gene on ApoE, assays were carried out to determinewhether ApoE expression correlates with human melanoma progression. Tothis end, published array-based expression data for ApoE (Haqq et al.,2005 Proc. Natl. Acad. Sci. USA 102, 6092-6097) was analyzed in nevi,primary, and metastatic lesions. Consistent with ametastasis-suppressive role, ApoE levels were significantly lower indistal organ metastases relative to primary (P<0.025) and nevi lesions(P<0.0003) (FIG. 5K).

Given its significant correlation with human melanoma progression, itwas next examined whether increasing ApoE signaling in melanoma cellscould have therapeutic efficacy in suppressing melanoma metastasis. Morespecifically, metastatic MeWo-LM2 cells were pre-incubated withrecombinant ApoE or BSA for 24 hours prior to injection into mice.Strikingly, pre-treatment of cancer cells with ApoE robustly suppressedmetastatic colonization by over 300-fold (FIG. 5L). This dramaticsuppression of metastasis by ApoE pre-incubation of melanoma cellsreflects that the effects of ApoE on melanoma cells are pivotal formetastatic initiation, as cells pre-treated with ApoE exhibit reducedinvasive ability, which is needed to initiate metastatic events leadingto lung colonization.

Given the robust influence exerted by ApoE on metastasis and metastaticphenotypes, as well as its strong association with human melanomaprogression, further assays were carried out to investigate the impactof genetic deletion of systemic ApoE on melanoma progression in animmunocompetent mouse model of melanoma metastasis. Consistent with amajor suppressive role for extracellular ApoE in metastasis, B16F10mouse melanoma cells injected into the circulation exhibited a greaterthan 7-fold increase in metastatic colonization in ApoE genetically nullmice compared to their wild-type littermates (FIG. 5M). These findingsestablish systemic and cancer-secreted ApoE as a robust suppressor ofhuman and mouse melanoma metastasis.

Example 7 Extracellular ApoE Divergently Targets Melanoma Cell LRPJ andEndothelial Cell LRP8 Receptors

In this example, assays were carried out to investigate the molecularmechanisms by which ApoE suppresses metastasis.

In order to identify the ApoE receptor(s) that mediate(s) invasion, downall four known ApoE receptors, VLDLR, LRP1, LRP8, and LDLR (Hatters etal., 2006 Trends Biochem. Sci. 31, 445-454; Hauser et al., 2011 Prog.Lipid Res. 50, 62-74) were knocked in melanoma cells. Interestingly,knock-down of LRP1, but not the other ApoE receptors, abolished the cellinvasion suppression effect induced by recombinant ApoE (FIG. 6A).Importantly, knock-down of LRP1 in metastatic LM2 cells, which displaylow levels of ApoE, only modestly increased cell invasion (FIG. 17A),suggesting the effects of LRP1 to be mediated by endogenous ApoE.

To determine if LRP1 also mediates the miRNA-dependent effects oninvasion and metastatic colonization, LRP1 was knocked down in thecontext of miRNA inhibition. LRP1 knock-down in the setting of miRNAsilencing rescued the suppressed invasion phenotype arising from miRNAinhibition (FIGS. 6B, 17B). Consistent with these in vitro results, LRP1knock-down significantly enhanced in vivo metastatic colonization by LM2cells silenced for miR-1908 (FIG. 6C, 17C). These findings reveal LRP1to be epistatically downstream of miRNA/ApoE-dependent melanoma invasionand metastatic colonization.

While the invasion phenotype reflects the cell-autonomous effects ofApoE on melanoma cells, the endothelial recruitment phenotype suggests anon-cell-autonomous role of cancer-expressed ApoE directly onendothelial cells. Consistent with this, pre-treatment of endothelialcells with ApoE significantly reduced their ability to migrate towardshighly metastatic cancer cells (FIG. 6D). In order to identify the ApoEreceptor(s) on endothelial cells that mediate(s) the endothelialrecruitment phenotype, all four known ApoE receptors were knocked downon endothelial cells. Interestingly, unlike for cancer cell invasion,knock-down of endothelial LRP8, but not any of the other receptors,selectively and significantly abrogated the inhibition of endothelialrecruitment caused by miRNA silencing (FIGS. 6E, 17D-E). These findingsare consistent with the LRP8 receptor being the downstream endothelialmediator of miRNA/ApoE-dependent effects on endothelial recruitment.

Next, assays were carried out to examine whether ApoE/LRP8 signalingmight also regulate general endothelial migration in a cancer cell-freesystem. Accordingly, antibody neutralization of ApoE, which is presentin endothelial cell media, significantly enhanced endothelial migration(FIG. 6F), while recombinant ApoE was sufficient to inhibit endothelialmigration in a trans-well assay (FIG. 6G) and a gradient-basedchemotactic assay (FIG. 6H) in an endothelial cell LRP8receptor-dependent manner. Importantly, addition of ApoE lead to adramatic (greater than 40-fold) suppression of VEGF-induced endothelialrecruitment in vivo into subcutaneous matrigel plugs (FIG. 6I).

Given the requirement and sufficiency of ApoE in mediating endothelialrecruitment, further assays were carried out to examine whether systemicApoE might regulate metastatic angiogenesis. Consistent with the robustsuppression of metastatic endothelial content by melanoma cell-secretedApoE (FIG. 4K), genetically null ApoE mice displayed higher blood vesseldensities within their lung metastatic nodules formed by B16F10 mousemelanoma cells compared to their wild-type littermates (FIG. 6J;2.41-fold increase, P=0.0055). Taken together, the above findings revealdual cell-autonomous/non-cell-autonomous roles for ApoE in metastasissuppression through divergent signaling mediated by melanoma cell LRP1and endothelial cell LRP8 receptors.

Example 8 MiR-199a-3p, miR-199a-5p, and miR-1908 as Robust Prognosticand Therapeutic Targets in Melanoma Metastasis

To examine whether the metastasis promoter miRNAs described herein couldserve as clinical predictors of metastatic outcomes, the expressionlevels of miR-199a-3p, miR-199a-5p, and miR-1908 were quantified in ablinded fashion by qRT-PCR in a cohort of human melanoma samplesobtained from patients at MSKCC. The relationships between the levels ofthese miRNAs in primary melanoma lesions and metastatic relapse outcomeswere then determined.

Importantly, patients whose primary melanoma lesions expressed higher(greater than the median for the population) levels of miR-199a-3p,miR-199a-5p, or miR-1908 were more likely to develop distal metastasesand exhibited significantly shorter metastasis-free survival times thanpatients whose primary melanomas expressed lower levels of each of thesemiRNAs (FIGS. 7A-C, P=0.0032 for miR-199a-3p, P=0.0034 for miR-199a-5p,and P=0.027 for miR-1908). Strikingly, the aggregate expression levelsof the three miRNAs displayed the strongest prognostic capacity instratifying patients at high risk from those with very low risk formetastatic relapse (FIG. 7D, P<0.0001). These clinical findings areconsistent with functional cooperativity between these miRNAs in theregulation of cancer progression and suggest utility for these moleculesas clinical prognostic biomarkers of melanoma metastasis.

In light of the current lack of effective treatment options for theprevention of melanoma metastasis and the strong prognostic value of thethree regulatory miRNAs in melanoma metastasis, these miRNAstherapeutically targeted using antisense LNA therapy (Elmer et al.,2008(a); Elmer et al., 2008(b)). Highly metastatic MeWo-LM2 cellspre-treated with LNA oligonucleotides antisense to mature miR-199a-3p,miR-199a-5p, or miR-1908 exhibited roughly a four-fold decrease inmetastatic activity. Given clinical evidence for cooperativity amongthese miRNAs, the impact of silencing all three miRNAs on metastaticprogression was examined. Remarkably, co-transfection of LNAs againstall three miRNAs suppressed metastatic colonization by overseventy-fold, revealing dramatic synergy and cooperativity betweenendogenous miR-199a-3p, miR-199a-5p, and miR-1908 (FIG. 7E, P=0.004).Importantly, inhibition of these miRNAs with triple LNA pre-treatmentdid not result in decreased in vitro proliferation (FIG. 18A),indicating that the dramatic metastasis suppression phenotype is notsecondary to impaired proliferation. Combinatorial LNA-mediated miRNAtargeting in the independent A375 metastatic derivative line alsosignificantly inhibited lung colonization (FIG. 18B).

Next, it was examined whether combinatorial LNA-induced miRNA inhibitioncould suppress systemic melanoma metastasis to multiple distant organs.Indeed, intracardiac injection of highly metastatic melanoma cellspre-treated with a cocktail of LNAs targeting the three regulatorymiRNAs revealed endogenous miR-199a-3p, miR-199a-5p, and miR-1908 topromote systemic melanoma metastasis (FIG. 7F). CombinatorialLNA-mediated inhibition of the three miRNAs lead to a reduction in thenumber of systemic metastatic foci (FIG. 7G) in distal sites such as thebrain and bone (FIGS. 7H-I).

Further assays were carried out to examine the therapeutic efficacy ofsystemically administered in vivo-optimized LNAs in melanoma metastasisprevention. To this end, highly metastatic MeWo-LM2 cells were injectedinto mice. The following day, mice were intravenously treated with LNAstargeting miR-199a-3p, miR-199a-5p, and miR-1908 at a low total dose(12.5 mg/kg) on a bi-weekly basis for four weeks. Notably, combinatorialLNA treatment reduced lung colonization by 9-fold (FIG. 7J, P=0.031)without any apparent signs of toxicity (FIG. 18C). Taken together, theabove findings reveal a novel miRNA-dependent regulatory network thatconverges on ApoE signaling to control cell-autonomous andnon-cell-autonomous features of melanoma metastatic progression (FIG.7K). The above basic studies have identified a set of miRNAs withpowerful prognostic and therapeutic potential in the clinical managementof melanoma.

Example 9 miRNA-Dependent Targeting of Apoe/LRP1 Signaling PromotesCancer Cell Invasion and Endothelial Recruitment Through CTGF Induction

In this example, Connective Tissue Growth Factor (CTGF) was identifiedas a down-stream mediator of ApoE/LRP1 signaling in cancer cell invasionand endothelial recruitment. CTGF expression level, as determined byqRT-PCR analysis and ELISA, is mediated by ApoE/LRP1 signaling (FIGS.8A, 8B, and 8C). Additionally, ApoE/LRP1 regulated cancer cell invasionand endothelial recruitment are mediated by CTGF (FIG. 8D, 8E).

Example 10 CTGF Mediates miRNA-Dependent Metastatic Invasion,Endothelial Recruitment, and Colonization

In this Examiner, assays were carried out to investigate whether CTGFmediates miRNA-dependent invasion and endothelial recruitment. Briefly,trans-well cell invasion and endothelial recruitment assays wereperformed on parental MeWo cells over-expressing miR-199a or miR-1908 inthe presence of a blocking antibody targeting CTGF. Indeed, it was foundthat mir-199a and mir-1908 dependent metastatic invasion and endothelialrecruitment are mediated by CTGF (FIGS. 9A and 9B). In order toinvestigate whether in vivo melanoma metastasis (metastaticcolonization) is mediated by CTGF, bioluminescence imaging was performedon lung metastasis by 5×104 parental MeWo cells knocked down for CTGF inthe setting of miR-199a or miR-1908 over-expression. Knock-down of CTGFin this setting resulted in significant reduction of in vivo melanomametastasis (FIG. 9C).

Example 11 Treatment with LXR Agonist GW3965 Elevates Melanoma Cell ApoEand DNAJA4 Levels and Suppresses Cancer Cell Invasion, EndothelialRecruitment, and Metastatic Colonization

Small molecule agonists of the Liver X Receptor (LXR) have previouslybeen shown to increase Apo E levels. To investigate whether increasingApo-E levels via LXR activation resulted in therapeutic benefit, assayswere carried out to assess the effect of the LXR agonist GW3965[chemical name:3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride) on Apo-E levels, tumor cell invasion,endothelial recruitment, and in vivo melanoma metastasis (FIG. 10).Incubation of parental MeWo cells in the presence of therapeuticconcentrations of GW3965 increased expression of ApoE and DNAJA4 (FIGS.10A and 10B). Pre-treatment of MeWO cells with GW3965 decreased tumorcell invasion (FIG. 10C) and endothelial recruitment (FIG. 10D). To testwhether GW3965 could inhibit metastasis in vivo, mice were administereda grain-based chow diet containing GW3965 (20 mg/kg) or a control diet,and lung metastasis was assayed using bioluminescence after tail-veininjection of 4×10⁴ parental MeWo cells into the mice (FIG. 10E). Oraladministration of GW3965 to the mice in this fashion resulted in asignificant reduction in in vivo melanoma metastasis (FIG. 10E).

Example 12 Identification of Mir-7 as an Endogenous Suppressor ofMelanoma Metastasis

In this example, miR-7 was identified as an endogenous suppressor ofmelanoma metastasis (FIG. 11). To test whether miR-7 suppresses melanomametastasis in vivo, its expression was knocked down in parental MeWocells using miR-Zip technology (FIG. 11A). Bioluminescence imaging plotof lung metastatic colonization following intravenous injection of 4×104parental MeWo cells expressing a short hairpin (miR-Zip) inhibitor ofmiR-7 (miR-7 KD) significantly increased lung metastasis in vivo (FIG.11A). Conversely, overexpression of miR-7 in LM2 cells significantlyreduced lung metastasis in vivo (FIG. 11B).

The complexity of cancer requires the application of systematic analyses(Pe'er and Hacohen, 2011). Via a systematic global approach, acooperative network of miRNAs was uncovered. The miRNAs are i)upregulated in highly metastatic human melanoma cells, ii) required andsufficient for metastatic colonization and angiogenesis in melanoma, andiii) robust pathologic predictors of human melanoma metastatic relapse.Through a transcriptomic-based and biologically guided targetidentification approach, miR-1908, miR-199a-3p, and miR-199a-5p werefound to convergently target the heat shock factor DNAJA4 and themetabolic gene ApoE. The requirement of each individual miRNA formetastasis indicates that these three convergent miRNAs arenon-redundant in promoting melanoma metastasis, while the robustsynergistic metastasis suppression achieved by combinatorial miRNAinhibition reveals functional cooperativity between these miRNAs,presumably achieved through maximal silencing of ApoE and DNAJA4. Theidentification of ApoE as a gene negatively regulated by threemetastasis promoter miRNAs, positively regulated by a metastasissuppressor gene (DNAJA4), and silenced in clinical metastasis sampleshighlights the significance of this gene as a suppressor of melanomaprogression.

Example 13 Identification of LXRβ Signaling as a Novel TherapeuticTarget in Melanoma

To identify nuclear hormone receptors that show broad expression inmelanoma, we examined the expression levels of all nuclear hormonereceptor family members across the NCI-60 collection of human melanomacell lines. Several receptors exhibited stable expression acrossmultiple melanoma lines, suggesting that they could represent novelpotential targets in melanoma (FIGS. 19A and 20A). Notably, out ofthese, liver-X receptors (LXRs) were previously shown to enhance ApoEtranscription in adipocytes and macrophages (Laffitte et al., 2001),while pharmacologic activation of RXRs was found to drive ApoEexpression in pre-clinical Alzheimer's models (Cramer et al., 2012).

Given the recently uncovered metastasis-suppressive role of ApoE inmelanoma (Pencheva et al., 2012), the ubiquitous basal expression ofLXRβ and RXRα in melanoma, and the availability of pharmacologic agentsto therapeutically activate LXRs and RXRs, we investigated whetheractivation of LXRs or RXRs in melanoma cells might inhibit melanomaprogression phenotypes. In light of the established roles of nuclearhormone receptors such as ER and AR in regulating breast and prostatecancer cell proliferation, we first examined whether pharmacologicagonism of LXRs or RXRs in melanoma cells affects in vitro cell growth.

Treatment of melanoma cells with two structurally-distinct LXR agonists,GW3965 2 or T0901317 1, or the RXR agonist bexarotene did not affectcell proliferation or cell viability rates (FIG. 20 B-C). We nextassessed the effects of LXR or RXR activation on cell invasion andendothelial recruitment—phenotypes displayed by metastatic melanoma andmetastatic breast cancer populations (Pencheva et al., 2012; Png et al.,2012). Treatment of the mutationally diverse MeWo (B-Raf/N-Raswild-type), HT-144 (B-Raf mutant), and SK-Mel-2 (N-Ras mutant) humanmelanoma lines as well as the SK-Mel-334.2 (B-Raf mutant) primary humanmelanoma line with GW3965 2 or T0901317 1 consistently suppressed theability of melanoma cells to invade through matrigel and to recruitendothelial cells in trans-well assays (FIG. 19B-C). In comparison,treatment with bexarotene suppressed invasion only in half of themelanoma lines tested and it did not significantly affect theendothelial recruitment phenotype (FIGS. 19B-C).

Given the superiority of LXR over RXR agonism in broadly inhibiting bothcell invasion and endothelial recruitment across multiple melanomalines, we investigated the requirement for LXR signaling in mediatingthe suppressive effects of LXR agonists. Knockdown of melanoma LXRβ, butnot LXRα, abrogated the ability of GW3965 2 and T0901317 1 to suppressinvasion and endothelial recruitment (FIG. 19D-G and FIGS. 20D-G),revealing melanoma-cell LXRβ to be the functional target of LXR agonistsin eliciting the suppression of these in vitro phenotypes. Our molecularfindings are consistent with LXRβ being the predominant LXR isoformexpressed by melanoma cells (FIG. 19A, P<0.0001).

The ubiquitous basal expression of LXRβ in melanoma is likely reflectiveof the general role that LXRs play in controlling lipid transport,synthesis, and catabolism (Calkin and Tontonoz, 2013). While such stableLXRβ expression would be key to maintaining melanoma cell metabolism andgrowth, it also makes LXR signaling an attractive candidate forbroad-spectrum therapeutic targeting in melanoma.

Example 14 Therapeutic Delivery of LXR Agonists Suppresses MelanomaTumor Growth

LXR agonists were originally developed as oral drug candidates for thepurpose of cholesterol lowering in patients with dyslipidemia andatherosclerosis (Collins et al., 2002; Joseph and Tontonoz, 2003). Thesecompounds were abandoned clinically secondary to their inability toreduce lipid levels in large-animal pre-clinical models (Groot et al.,2005).

Given the robust ability of GW3965 2 and T0901317 1 to suppress in vitromelanoma progression phenotypes (FIG. 19B-C), we investigated whethertherapeutic LXR activation could be utilized for the treatment ofmelanoma. Indeed, oral administration of GW3965 2 or T0901317 1 at lowdoses (20 mg/kg), subsequent to formation of subcutaneous tumorsmeasuring 5-10 mm3 in volume, suppressed tumor growth by the aggressiveB16F10 mouse melanoma cells in an immunocompetent model by 67% and 61%,respectively (FIG. 21A-B). Administration of a higher LXR agonist dose(100 mg/kg) led to an 80% reduction in tumor growth (FIG. 21A),consistent with dose-dependent suppressive effects.

Oral administration of GW3965 2 also robustly suppressed tumor growth bythe MeWo (70% inhibition) and SK-Mel-2 (49% inhibition) human melanomacell lines, as well as the SK-Mel-334.2 primary human melanoma line (73%inhibition) (FIG. 21C-E and FIG. 22A).

Encouraged by the robust tumor-suppressive impact of LXR agonists onsmall tumors (5-10 mm3) (FIG. 21A-E), we next investigated whether LXRactivation therapy could inhibit the growth of large (˜150 mm3) tumors.

We found that treatment with GW3965 2 led to a roughly 50% reduction inthe growth of established large B16F10 tumors (FIG. 21F). Importantly,therapeutic delivery of GW3965 2 subsequent to tumor establishmentsubstantially prolonged the overall survival time of immunocompetentmice injected with mouse B16F10 cells, immunocompromised mice bearingtumor xeongrafts derived from the human MeWo established melanoma line,as well as the SK-Mel.334-2 primary human melanoma line (FIG. 21G-I).These findings are consistent with broad-spectrum responsiveness to LXRactivation therapy across melanotic and amelanotic established melanomatumors of diverse mutational subtypes: B-Raf and N-Ras wild-type (B16F10and MeWo; FIG. 21A-C), B-Raf mutant (SK-Mel-334.2; FIG. 21D), and N-Rasmutant (SK-Mel-2; FIG. 21E).

We next sought to determine the cell biological phenotypes regulated byLXR agonists in suppressing tumor growth. Consistent with the inhibitoryeffects of GW3965 2 on endothelial recruitment by melanoma cells invitro, GW3965 2 administration led to a roughly 2-fold reduction in theendothelial cell content of tumors (FIG. 21J). This effect wasaccompanied by a modest decrease (23%) in the number of activelyproliferating tumor cells in vivo (FIG. 21K) without a change in thenumber of apoptotic cells (FIG. 21L). These results suggest that, inaddition to reducing local tumor invasion, LXR activation suppressesmelanoma tumor growth primarily through inhibition of tumor angiogenesiswith a resulting reduction in in vivo proliferation.

Example 15 LXR Agonism Suppresses Melanoma Metastasis to the Lung andBrain and Inhibits the Progression of Incipient Metastases

The strong suppressive effects of LXR agonists on melanoma tumor growthmotivated us to examine whether LXR activation could also suppressmetastatic colonization by melanoma cells. To this end, pre-treatment ofhuman MeWo melanoma cells with GW3965 2 led to a more than 50-foldreduction in their metastatic colonization capacity (FIG. 23A). In lightof this dramatic inhibitory effect, we next assessed the ability oforally administered LXR agonists to suppress metastasis.Immunocompromised mice that were orally administered GW3965 2 orT0901317 1 experienced 31-fold and 23-fold respective reductions in lungmetastatic colonization by human MeWo cells (FIG. 23B-C). Treatment withGW3965 2 also suppressed metastatic colonization by the HT-144 melanomaline (FIG. 23D) as well as the SK-Mel-334.2 primary melanoma line (FIG.23E).

GW3965 2 is a lipophilic molecule that can efficiently cross the bloodbrain barrier and potently activate LXR signaling in the brain.Consistent with this, oral delivery of GW3965 2 was previously shown toimprove amyloid plaque pathology and memory deficits in pre-clinicalmodels of Alzheimer's disease (Jiang et al., 2008). We thus wonderedwhether LXR agonism could exhibit therapeutic activity in thesuppression of melanoma brain metastasis—a dreaded melanoma outcome indire need of effective therapies (Fonkem et al., 2012). Notably, oraladministration of GW3965 2 inhibited both systemic dissemination andbrain colonization following intracardiac injection of brain-metastaticmelanoma cells derived from the MeWo parental line (FIG. 23F). Theseresults reveal robust metastasis suppression by LXR activation therapyacross multiple melanoma lines and in multiple distal organ metastaticsites.

Encouraged by the robust effects observed in suppressing metastasisformation (FIG. 23A-F), we next sought to determine whether LXRactivation therapy could halt the progression of melanoma cells that hadalready metastatically disseminated. We first tested the ability ofGW3965 2 to reduce lung colonization by melanoma cells disseminatingfrom an orthotopic site following removal of the primary tumor (FIG.23G). Importantly, oral administration of GW3965 2 post-tumor excisioninhibited lung colonization by disseminated melanoma cells by 17-fold(FIG. 23H). Remarkably, treatment of mice with GW3965 2 alsodramatically suppressed (28-fold) colonization by incipient lungmetastases that had progressed 8-fold from the baseline at seeding (FIG.23I). Consistent with LXR activation inhibiting metastatic initiation,GW3965 2 treatment decreased the number of macroscopic metastaticnodules formed (FIG. 23J). Finally, treatment of mice with GW3965 2 inthis ‘adjuvant’ pre-clinical context significantly prolonged theirsurvival times following metastatic colonization (FIG. 23K).

Example 16 LXR Activation Reduces Melanoma Progression and Metastasis ina Genetically-Driven Mouse Model of Melanoma

Roughly 60% of human melanoma tumors are marked by activating mutationsin the Braf oncogene, with one single amino acid variant, B-RafV600E,being the predominant mutation found (Davies et al., 2002). Nearly 20%of melanomas exhibit activating mutations in B-Raf with concurrentsilencing of the Pten tumor-suppressor, which drives progression to amalignant melanoma state (Tsao et al., 2004; Chin et al., 2006).Recently, Tyrosinase (Tyr)-driven conditional B-Raf activation and Ptenloss were shown to genetically cooperate in driving mouse melanomaprogression (Dankort et al., 2009).

To determine whether LXR activation could suppress melanoma progressionin this genetically-initiated model, we induced melanomas in Tyr::CreER;B-RafV600E/+; Ptenlox/+ and Tyr::CreER; B-RafV600E/+; Ptenlox/lox miceby intraperitoneal administration of 4-hydroxytamoxifen (4-HT). Notably,oral administration of GW3965 2 following melanoma initiation attenuatedtumor progression and significantly extended the overall survival timesof both PTEN heterozygous Tyr::CreER; B-RafV600E/+; Ptenlox/+ and PTENhomozygous Tyr::CreER; B-RafV600E/+; Ptenlox/lox mice (FIG. 24A-B andFIG. 25A-B). Next, we examined the ability of GW3965 2 to suppressmelanoma metastasis in this genetic context. While we did not detectmacroscopic metastases in the lungs or brains of 4-HT-treatedTyr::CreER; B-Raf^(V600E/+); Pten^(lox/lox) control mice, weconsistently observed melanoma metastases to the salivary gland lymphnodes. Importantly, Tyr::CreER; B-Raf^(V600E/+); Pten^(lox/lox) micetreated with GW3965 2 exhibited a decrease in the number of lymphaticmetastases detected post-mortem (FIG. 24C). These findings indicate thatLXR activation inhibits orthotopic metastasis in a genetically-drivenmelanoma model, in addition to its suppressive effects on primarymelanoma tumor progression.

The cooperativity between B-Raf activation and Pten loss in drivingmelanoma progression can be further enhanced by inactivation of CDKN2A,a cell cycle regulator frequently mutated in familial melanomas(Hussussian et al., 1994; Kamb et al., 1994). We thus examined theeffect of LXR activation on B-Raf^(V600E/+); Pten^(−/−); CDKN2A^(−/−)melanomas, allowing us to test the therapeutic efficacy of LXR agonismin a more aggressive genetically-driven melanoma progression model.Importantly, therapeutic delivery of GW3965 2 robustly inhibited tumorgrowth and lung metastasis by B-Raf^(V600E/+); Pten^(−/−); CDKN2A^(−/−)primary mouse melanoma cells injected into syngeneic immunocompetentmice and extended the overall survival of mice bearing B-Raf^(V600E/+);Pten^(−/−); CDKN2A^(−/−) melanoma burden (FIG. 24D-F). Taken together,the robust suppression of melanoma progression across independentxenograft and genetically-induced immunocompetent melanoma mouse modelsthat exhibit the diverse mutational profiles of human melanomasmotivates the clinical testing of LXR activation therapy.

Example 17 Pharmacologic Activation of LXRβ Suppresses MelanomaPhenotypes by Transcriptionally Inducing Melanoma-Cell ApoE Expression

We next sought to determine the downstream molecular target of LXRβ thatmediates suppression of melanoma progression. To this end, wetranscriptomically profiled human MeWo melanoma cells treated with theLXR agonist GW3965 2.

Out of the 365 genes that were significantly induced in response to LXRactivation, we identified ApoE, a previously validated transcriptionaltarget of LXRs in macrophages and adipocytes (Laffitte et al., 2001), asthe top upregulated secreted factor in melanoma cells (FIG. 26).Quantitative real-time PCR (qRT-PCR) validation revealed robustupregulation of ApoE transcript expression following treatment withindependent LXR agonists across multiple human melanoma lines (FIG.27A-C).

In light of the previously reported metastasis-suppressive function ofApoE in melanoma (Pencheva et al., 2012), we investigated whether LXRβactivation suppresses melanoma progression through transcriptionalinduction of ApoE. Indeed, GW3965 2 and T0901317 1 were found to enhancethe melanoma cell-driven activity of a luciferase reporter constructcontaining the ApoE promoter fused to either of two previouslycharacterized LXR-binding multi-enhancer elements (ME.1 or ME.2)(Laffitte et al., 2001) (FIG. 28A). Importantly, this transcriptionalinduction resulted in elevated levels of secreted ApoE protein (FIG.28B). Consistent with direct LXRβ targeting of ApoE in melanoma cells,neutralization of extracellular ApoE with an antibody fully blocked theLXRβ-mediated suppression of cell invasion and endothelial recruitmentand further enhanced these phenotypes relative to the control IgGtreatment (FIG. 28C-G and FIG. 27D-F), revealing the effects of LXRagonism to be modulated by extracellular ApoE.

Additionally, molecular knockdown of ApoE in melanoma cells also blockedthe GW3965 2-mediated suppression of cell invasion and endothelialrecruitment phenotypes (FIG. 27G-H). In agreement with this, melanomacell depletion of LXRβ, but not LXRa, abrogated the ability of GW3965 2and T0901317 1 to upregulate ApoE transcription and ultimately proteinexpression (FIG. 28H-I and FIG. 27I-K). Collectively, these findingsindicate that pharmacologic activation of LXRβ, the predominant LXRisoform expressed by melanoma cells, suppresses cell-intrinsic invasionand endothelial recruitment by melanoma cells through transcriptionallyactivating ApoE expression in melanoma cells.

Example 18 Engagement of Melanoma-Derived and Systemic ApoE by LXRβActivation Therapy

The LXRβ-induced suppression of key melanoma phenotypes by extracellularApoE in vitro suggested that the suppressive effects of LXR agonists invivo might be further augmented by the activation of LXRs in peripheraltissues, which could serve as robust sources of extracellular ApoE.

Importantly, such non-transformed tissues would be less vulnerable todeveloping resistance to LXR activation therapy, allowing for chronicApoE induction in patients. We thus investigated whether therapeutic LXRagonism suppresses melanoma progression by inducing ApoE derived frommelanoma cells or systemic tissues. Consistent with LXRβ agonismincreasing ApoE expression in melanoma cells in vivo, ApoE transcriptlevels were upregulated in melanoma primary tumors as well as inmelanoma lung and brain metastases dissociated from mice that were fedan LXR agonist-supplemented diet (FIG. 29A-E). Importantly, treatment ofmice with either GW3965 2 or T0901317 1 significantly elevated ApoEprotein expression in systemic adipose, lung, and brain tissues of mice(FIGS. 30A-B) and also upregulated ApoE transcript levels in circulatingwhite blood cells (FIG. 30C). These results indicate that LXR activationtherapy induces both melanoma-cell and systemic tissue ApoE expressionin vivo.

To determine the in vivo requirement of melanoma-derived and systemicLXR activation for the tumor-suppressive effects of orally administeredLXR agonists, we first tested the ability of GW3965 2 to suppress tumorgrowth by B16F10 mouse melanoma cells depleted of LXRβ.

Consistent with our findings in human melanoma cells, knockdown of mousemelanoma-cell LXRβ abrogated the GW3965-mediated induction of ApoEexpression (FIG. 29F-H). Despite this, melanoma-cell LXRβ knockdown wasunable to prevent the suppression of tumor growth by GW3965 2 (FIG.29D), implicating a role for systemic LXR activation in tumor growthinhibition by GW3965 2. To identify the LXR isoform that mediates thisnon-tumor autonomous suppression of melanoma growth by LXR agonists, weexamined the effects of GW3965 2 on tumors implanted onto LXRa or LXRβgenetically null mice. Interestingly, genetic ablation of systemic LXRβblocked the ability of GW3965 to suppress melanoma tumor growth, whileLXRa inactivation had no effect on tumor growth inhibition by GW3965(FIG. 6D). Importantly, the upregulation of systemic ApoE expression byGW3965 2, an agonist with 6-fold greater activity towards LXRβ thanLXRa, was abrogated in LXRβ−/−, but not in LXRα−/− mice (FIG. 30E andFIG. 29I). These results indicate that ApoE induction by GW3965 2 inperipheral tissues is predominantly driven by systemic LXRβ activation.In agreement with this, we find systemic LXRβ to be the primarymolecular target and effector of GW3965 2 in mediating melanoma tumorgrowth suppression.

We next examined whether ApoE is required for the in vivomelanoma-suppressive effects of LXR agonists. Consistent with the lackof an impact for melanoma-cell LXRβ knockdown on the tumor-suppressiveactivity of GW3965 2, depletion of melanoma-cell ApoE did not preventtumor growth inhibition by GW3965 2 neither (FIG. 29F-H and FIG. 30F).These findings suggest that the tumor suppressive effects of GW3965 2might be primarily mediated through ApoE induction in systemic tissues.

Indeed, GW3965 2 was completely ineffective in suppressing tumor growthin mice genetically inactivated for ApoE (FIG. 30F), revealing systemicApoE as the downstream effector of systemic LXRβ in driving melanomatumor growth suppression. Interestingly, in contrast to primary tumorgrowth regulation, knockdown of melanoma-cell ApoE partially preventedthe metastasis-suppressive effect of GW3965 2 (FIG. 30G). Similarly,genetic inactivation of ApoE only partially prevented the metastasissuppression elicited by GW3965 2 as well (FIG. 30G). The GW3965-driveninhibition of metastasis was completely blocked only in the context ofboth melanoma-cell ApoE knockdown and genetic inactivation of systemicApoE (FIG. 30G), indicative of a requirement for both melanoma-derivedand systemic ApoE engagement by LXRβ in suppressing metastasis. We thusconclude that the effects of LXRβ activation on primary tumor growth areelicited primarily through systemic ApoE induction, while the effects ofLXRβ agonism on metastasis are mediated through ApoE transcriptionalinduction in both melanoma cells and systemic tissues.

The identification of ApoE as the sole downstream mediator of theLXRβ-induced suppression of melanoma phenotypes further highlights theimportance of this gene as a suppressor of melanoma progression. Todetermine whether ApoE expression is clinically prognostic of melanomametastatic outcomes, we assessed ApoE protein levels by performingblinded immunohistochemical analysis on 71 surgically resected humanprimary melanoma lesions.

We found that patients whose melanomas had metastasized exhibitedroughly 3-fold lower ApoE expression in their primary tumors relative topatients whose melanomas did not metastasize (FIG. 30H, P=0.002).Remarkably, ApoE expression levels in patients' primary melanoma lesionsrobustly stratified patients at high risk from those at low risk formetastatic relapse (FIG. 30I, P=0.002). These observations areconsistent with previous findings that revealed significantly lowerlevels of ApoE in distant melanoma metastases relative to primarylesions (Pencheva et al., 2012). Collectively, this work indicates thatApoE, as a single gene, could likely act as a prognostic and predictivebiomarker in primary melanomas to identify patients that i.) are at riskfor melanoma metastatic relapse and as such ii.) could obtain clinicalbenefit from LXRβ agonist-mediated ApoE induction.

Example 19 LXR/3 Activation Therapy Suppresses the Growth of MelanomasResistant to Dacarbazine and Vemurafenib

Encouraged by the robust ability of LXRβ activation therapy to suppressmelanoma tumor growth and metastasis across a wide range of melanomalines of diverse mutational backgrounds, we next sought to determinewhether melanomas that are resistant to two of the mainstay clinicalagents used in the management of metastatic melanoma—dacarbazine andvemurafenib—could respond to LW-activation therapy.

To this end, we generated B16F10 clones resistant to dacarbazine (DTIC)by continuously culturing melanoma cells in the presence of DTIC for twomonths. This yielded a population of cells that exhibited a 7-foldincrease in viability in response to high-dose DTIC treatment comparedto the parental B16F10 cell line (FIG. 31A). To confirm that this invitro-derived DTIC clone was also resistant to DTIC in vivo, we assessedthe effects of dacarbazine treatment on tumor growth.

While dacarbazine significantly suppressed the growth of theDTIC-sensitive parental line (FIG. 31B), it did not affect tumor growthby B16F10 DTIC-resistant cells (FIG. 31C). GW3965 2 robustly suppressedtumor growth by the DTIC-resistant B16F10 melanoma clone by more than70% (FIGS. 31C-D). Importantly, oral delivery of GW3965 2 also stronglyinhibited the growth of in vivo-derived DTIC-resistant human melanomatumors formed by the independent MeWo cell line (FIG. 31E-F and FIG.32A).

These results reveal that LXRβ agonism is effective in suppressingmultiple melanoma cell populations that are resistant to dacarbazine—theonly FDA-approved cytotoxic chemotherapeutic in metastatic melanoma. Ourfindings have important clinical implications for melanoma treatmentsince all stage IV patients who are treated with dacarbazine ultimatelyprogress by developing tumors that are resistant to this agent.

We tested the impact of LXRβ activation therapy on melanoma cellsresistant to the recently approved B-Raf kinase inhibitor, vemurafenib—aregimen that shows activity against B-Raf-mutant melanomas (Bollag etal., 2010; Sosman et al., 2012). Numerous investigators have derivedmelanoma lines resistant to vemurafenib (Poulikakos et al., 2011; Shi etal., 2012, Das Thakur et al., 2013). GW3965 2 treatment suppressed thegrowth of the previously derived SK-Mel-239 vemurafenib-resistant lineby 72% (FIG. 31G) and significantly prolonged the survival of micebearing vemurafenib-resistant melanoma burden (FIG. 31H). Our findingsfrom combined pharmacologic, molecular and genetic studies in diversepre-clinical models of melanoma establish LXRβ targeting as a noveltherapeutic approach that robustly suppresses melanoma tumor growth andmetastasis through the transcriptional induction of ApoE—a keysuppressor of melanoma invasion and metastatic angiogenesis (Pencheva etal., 2012; FIG. 31I).

Example 20 Treatment with ApoE Inhibits Tumor Cell Invasion andEndothelial Recruitment Across Multiple Cancer Types, Including BreastCancer, Renal Cell Cancer and Pancreatic Cancer

In order to determine if ApoE treatment could be effective for treatingcancer types in addition to melanoma, in vitro assays were performed toassess the effect of ApoE treatment on several different cancer celllines, including breast cancer, renal cell cancer, and pancreatic cancercell lines (FIG. 33).

The ability of cancer cells to invade through matrigel in vitro wastested by using a trans-well matrigel invasion chamber system (354480,BD Biosciences). Various cancer cell lines were serum-starved overnightin media containing 0.2% FBS. The following day, invasion chambers werepre-equilibrated prior to the assay by adding 0.5 mL of starvation mediato the top and bottom wells. Meanwhile, cancer cells were trypsinizedand viable cells were counted using the trypan blue dead cell exclusiondye. Cancer cells were then resuspended at a concentration of 1×105cells/1 mL starvation media, and 0.5 mL of cell suspension, containing5×104 cells, was seeded into each trans-well. To determine the effect ofrecombinant ApoE on cancer cell invasion, human recombinant ApoE3 (4696,Biovision) or BSA were added to each trans-well at 100 μg/mL at thestart of the assay. The invasion assay was allowed to proceed for 24hours at 37° C. Upon completion of the assay, the inserts were washed inPBS, the cells that did not invade were gently scraped off from the topside of each insert using q-tips, and the cells that invaded into thebasal insert side were fixed in 4% PFA for 15 minutes at roomtemperature. Following fixation, the inserts were washed in PBS and thencut out and mounted onto slides using VectaShield mounting mediumcontaining DAPI nuclear stain (H-1000, Vector Laboratories). The basalside of each insert was imaged using an inverted fluorescence microscope(Zeiss Axiovert 40 CFL) at 5× magnification, and the number ofDAPI-positive cells was quantified using ImageJ.

Indeed, treatment with ApoE inhibited both tumor cell invasion andendothelial recruitment across all three of these cancer types (FIG.33A-I).

Example 21 LXR Agonists LXR-623, WO-2007-002563 Ex. 19, WO-2010-0138598Ex. 9, and SB742881, Induce ApoE Expression in Human Melanoma Cells

Given that ApoE activation by treatment with LXR agonists GW3965 2 andT0901317 1 resulted in therapeutic benefit for inhibiting tumor growthand metastasis, we next examined the ability of other LXR agonists toinduce ApoE expression in human melanoma cell lines (FIG. 34).

To determine the effect of the various LXR agonists (LXR-623,WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, and SB742881 on ApoEexpression in melanoma cells, 1×105 human MeWo melanoma cells wereseeded in a 6-well plate. The following day, DMSO or the respective LXRagonist was added to the cell media at a concentration of 500 nM, 1 μM,or 2 μM, as indicated, and the cells were incubated in the presence ofDMSO or the drug for 48 hours at 37° C. The total amount of DMSO addedto the cell media was kept below 0.2%. RNA was extracted from whole celllysates using the Total RNA Purification Kit (17200, Norgen). For everysample, 600 ng of RNA was reverse transcribed into cDNA using the cDNAFirst-Strand Synthesis kit (Invitrogen). Approximately 200 ng of cDNAwas mixed with SYBR® green PCR Master Mix and the corresponding forwardand reverse primers specific for detection of human ApoE. Each reactionwas carried out in quadruplicates, and ApoE mRNA expression levels weremeasured by quantitative real-time PCR amplification using an ABI Prism7900HT Real-Time PCR System (Applied Biosystems). The relative ApoEexpression was determined using the ΔΔCt method. GAPDH was used as anendogenous control for normalization purposes.

Indeed, treatment with the LXR agonists LXR-623, WO-2007-002563 Ex. 19,WO-2010-0138598 Ex. 9, and SB742881 all led to varied degrees of ApoEexpression induction. (FIG. 34A-C).

Example 22 Treatment with the LXR Agonist GW3965 Inhibits In Vitro TumorCell Invasion of Renal Cancer, Pancreatic Cancer, and Lung Cancer

We have demonstrated that treatment with LXR agonists resulted ininhibition of melanoma tumor cell invasion. Given that this effect ismediated by activation of ApoE expression, we hypothesized thattreatment with LXR agonists would result in inhibition of in vitro tumorcell invasion in breast cancer, pancreatic cancer, and renal cancer,since these cancer types were responsive to ApoE treatment. In order totest this hypothesis, we performed in vitro tumor cell invasion assaysby treating breast cancer, pancreatic cancer, and renal cell cancer celllines with the LXR agonist GW3965 2 (FIG. 35).

Various cell lines (5×10⁴ RCC human renal cancer cells, 5×10⁴ PANC1human pancreatic cancer cells, and 5×10⁴ H460 human lung cancer cells)were treated with DMSO or GW3965 at 1 μM for 56 hours. The cells wereserum starved for 16 hours in 0.2% FBS media in the presence of DMSO orGW3965. Following serum starvation, the cells were subjected to thetrans-well invasion assay using a matrigel invasion chamber system(354480, BD Biosciences). Invasion chambers were pre-equilibrated priorto the assay by adding 0.5 mL of starvation media to the top and bottomwells. Meanwhile, cancer cells were trypsinized and viable cells werecounted using trypan blue. Cancer cells were then resuspended at aconcentration of 1×10⁵ cells/1 mL starvation media, and 0.5 mL of cellsuspension, containing 5×10⁴ cells, was seeded into each trans-well. Theinvasion assay was allowed to proceed for 24 hours at 37° C. Uponcompletion of the assay, the inserts were washed in PBS, the cells thatdid not invade were gently scraped off from the top side of each insertusing q-tips, and the cells that invaded into the basal insert side werefixed in 4% PFA for 15 minutes at room temperature. Following fixation,the inserts were washed in PBS and then cut out and mounted onto slidesusing VectaShield mounting medium containing DAPI nuclear stain (H-1000,Vector Laboratories). The basal side of each insert was imaged using aninverted fluorescence microscope (Zeiss Axiovert 40 CFL) at 5×magnification, and the number of DAPI-positive cells was quantifiedusing ImageJ.

Indeed, treatment with GW3965 2 resulted in inhibition of tumor cellinvasion in all three cancer types tested (FIG. 35A-C). This furtherdemonstrated the broad therapeutic potential of LXR agonists fortreating various cancer types.

Example 23 Treatment with the LXR Agonist GW3965 Inhibits Breast CancerTumor Growth In Vivo

We have demonstrated that LXR agonists inhibit in vitro cancerprogression phenotypes in breast cancer, pancreatic cancer, and renalcancer. To investigate if LXR agonist treatment inhibits breast cancerprimary tumor growth in vivo, mice injected with MDA-468 human breastcancer cells were treated with either a control diet or a dietsupplemented with LXR agonist GW3965 2 (FIG. 36).

To determine the effect of orally delivered GW3965 2 on breast cancertumor growth, 2×10⁶ MDA-468 human breast cancer cells were resuspendedin 50 μL PBS and 50 μL matrigel and the cell suspension was injectedinto both lower memory fat pads of 7-week-old Nod Scid gamma femalemice. The mice were assigned to a control diet treatment or aGW3965-supplemented diet treatment (75 mg/kg/day) two days prior toinjection of the cancer cells. The GW3965 2 drug compound was formulatedin the mouse chow by Research Diets, Inc. Tumor dimensions were measuredusing digital calipers, and tumor volume was calculated as (smalldiameter) x (large diameter)/2.

Treatment with GW3965 resulted in significant reduction in breast cancertumor size in vivo (FIG. 36).

Example 24 Effects of Treatment with LXR Agonists LXR-623,WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, and SB742881 on In VitroMelanoma Progression Phenotypes

We have demonstrated the ability of various LXR agonists to induce ApoEexpression with varying potency in melanoma cells (FIG. 34). Since thetherapeutic effect of LXR agonists on cancer is via activation of ApoEexpression, we hypothesized that the therapeutic potency of any givenLXR agonist is directly correlated with its ability to induce ApoEexpression. To confirm this, we quantified the effect of treatment withvarious LXR agonists on in vitro endothelial recruitment and tumor cellinvasion of melanoma cells. As shown in FIG. 37, the degree to which LXRagonists inhibit in vitro cancer progression phenotypes is related tothe LXR agonist's ApoE induction potency.

Cell Invasion: MeWo human melanoma cells were treated with DMSO,LXR-623, WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, or SB742881 at 1μM each for 56 hours. The cells were then serum starved for 16 hours in0.2% FBS media in the presence of each corresponding drug or DMSO.Following serum starvation, the cells were subjected to the trans-wellinvasion assay using a matrigel invasion chamber system (354480, BDBiosciences). Invasion chambers were pre-equilibrated prior to the assayby adding 0.5 mL of starvation media to the top and bottom wells.Meanwhile, cancer cells were trypsinized and viable cells were countedusing trypan blue. Cancer cells were then resuspended at a concentrationof 2×10⁵ cells/1 mL starvation media, and 0.5 mL of cell suspension,containing 1×10⁵ cells, was seeded into each trans-well. The invasionassay was allowed to proceed for 24 hours at 37° C. Upon completion ofthe assay, the inserts were washed in PBS, the cells that did not invadewere gently scraped off from the top side of each insert using q-tips,and the cells that invaded into the basal insert side were fixed in 4%PFA for 15 minutes at room temperature. Following fixation, the insertswere washed in PBS, cut out, and mounted onto slides using VectaShieldmounting medium containing DAPI nuclear stain (H-1000, VectorLaboratories). The basal side of each insert was imaged using aninverted fluorescence microscope (Zeiss Axiovert 40 CFL) at 5×magnification, and the number of DAPI-positive cells was quantifiedusing ImageJ.

Endothelial Recruitment:

MeWo human melanoma cells were treated with DMSO, LXR-623,WO-2007-002563 Ex. 19, WO-2010-0138598 Ex. 9, or SB742881 at 1 μM eachfor 56 hours. Subsequently, 5×10⁴ cancer cells were seeded into 24-wellplates in the presence of each drug or DMSO and allowed to attach for 16hours prior to starting the assay. Human umbilical vein endothelialcells (HUVEC cells) were serum-starved in 0.2% FBS-containing mediaovernight. The following day, 1×10⁵ HUVEC cells were seeded into a 3.0μm HTS Fluoroblock insert (351151, BD Falcon) fitted into each wellcontaining the cancer cells at the bottom. The HUVEC cells were allowedto migrate towards the cancer cells for 20 hours, after which theinserts were washed in PBS, fixed in 4% PFA, labeled with DAPI, andmounted on slides. The basal side of each insert was imaged using aninverted fluorescence microscope (Zeiss Axiovert 40 CFL) at 5×magnification, and the number of DAPI-positive cells was quantifiedusing ImageJ.

LXR agonists that potently induce ApoE expression (e.g. WO-2010-0138598Ex. 9 and SB742881) are more effective at inhibiting cancer progressionphenotypes (FIG. 37) than lower potency LXR agonists. This furtherdemonstrates that the therapeutic benefit of LXR agonist treatment forcancer is a result of ApoE induction.

Example 25 Treatment with LXR Agonists Inhibit Melanoma Tumor Growth InVivo

We have demonstrated that LXR agonists that induce ApoE expressioninhibit in vitro tumor activity. To confirm if these agonists inhibitmelanoma tumor growth in vivo, mice that were injected with B16F10melanoma cells were treated with either LXR-623, WO-2007-002563 Ex. 19,WO-2010-0138598 Ex. 9, or SB742881.

To assess the effect of orally administered LXR-623, WO-2007-002563 Ex.19, WO-2010-0138598 Ex. 9, or SB742881 on melanoma tumor growth, 5×10⁴B16F10 mouse melanoma cells were resuspended in 50 μL PBS and 50 μLmatrigel and the cell suspension was subcutaneously injected into bothlower dorsal flanks of 7-week-old C57BL/6 mice. The mice were palpateddaily for tumor formation and after detection of tumors measuring 5-10m³ in volume, the mice were assigned to a control chow or a chowcontaining each respective LXR agonist: LXR-623 (20 mg/kg/day),WO-2007-002563 Ex. 19 (100 mg/kg/day), WO-2010-0138598 Ex. 9 (10mg/kg/day or 100 mg/kg/day), or SB742881 (100 mg/kg/day). The LXR drugcompounds were formulated in the mouse chow by Research Diets, Inc.Tumor dimensions were measured using digital calipers, and tumor volumewas calculated as (small diameter) x (large diameter)/2.

Consistent with our in vitro data, LXR agonists that potently induceApoE expression in vitro (WO-2010-0138598 Ex. 9, and SB742881)significantly inhibited melanoma primary tumor growth in vivo (FIG. 38).This is also consistent with our results demonstrating that other LXRagonists which potently induce ApoE expression (GW3965 2, T0901317 1)also inhibit primary tumor growth in vivo (FIG. 21).

Accordingly, the above examples focused on characterizing the molecularand cellular mechanisms by which it exerts its effects. To this end, itwas found that ApoE targets two distinct, yet homologous, receptors ontwo diverse cell types. ApoE acting on melanoma cell LRP1 receptorsinhibits melanoma invasion, while its action on endothelial cell LRP8receptors suppresses endothelial migration. The results fromloss-of-function, gain-of-function, epistasis, clinical correlation, andin vivo selection derivative expression analyses give rise to a modelwherein three miRNAs convergently target a metastasis suppressor networkto limit ApoE secretion, thus suppressing ApoE/LRP1 signaling onmelanoma cells and ApoE/LRP8 signaling on endothelial cells (FIG. 7K).Although the above systematic analysis has identified ApoE and DNAJA4 askey targets and direct mediators of the metastatic phenotypes regulatedby these miRNAs, it cannot be excluded that the three miRNAs mayindividually retain additional target genes whose silencing maycontribute to metastatic progression. The ability of ApoE or DNAJA4knock-down to fully rescue the metastasis suppression phenotypes seenwith individual miRNA silencing, however, strongly suggests that thesegenes are the key mediators of the miRNA-dependent effects onmetastasis.

The above results reveal combined molecular, genetic, and in vivoevidence for a required and sufficient role for ApoE in the suppressionof melanoma metastatic progression. ApoE can distribute in thecirculatory system both in a lipoprotein-bound and a lipid-free state(Hatters et al., 2006). While it has been shown that lipid-freerecombinant ApoE is sufficient to suppress melanoma invasion andendothelial migration, it is possible that ApoE contained in lipoproteinparticles could also suppress melanoma invasion and endothelialrecruitment. The ability of recombinant ApoE to inhibit thesepro-metastatic phenotypes, as well as the increased melanoma invasionand endothelial recruitment phenotypes seen with antibody-mediated ApoEneutralization suggests that the ApoE molecule itself is the keymediator of these phenotypes. Consistent with the findings disclosedherein, a synthetic peptide fragment of ApoE was previously found toinhibit endothelial migration through unknown mechanisms (Bhattacharjeeet al., 2011). The findings disclosed herein are consistent with a rolefor melanoma cell-secreted and systemic endogenous ApoE in inhibitingendothelial recruitment, which is not secondary to impaired endothelialcell growth.

The above-described molecular, genetic, and in vivo studies reveal arole for endogenous cancer-derived ApoE in the modulation of endothelialmigration and cancer angiogenesis through endothelial LRP8 receptorsignaling. This robust non-cell-autonomous endothelial recruitmentphenotype mediated by ApoE/LRP8 signaling suggests that ApoE may alsomodulate metastatic angiogenesis in other cancer types, and such ageneral role for ApoE in cancer angiogenesis biology remains to beexplored. ApoE is a polymorphic molecule with well-established roles inlipid, cardiovascular, and neurodegenerative disorders. Its three majorvariants, ApoE2, ApoE3, and ApoE4, display varying representations inthe human population, with ApoE3 being the most common variant (Hatterset al., 2006). The three isoforms differ at residues 112 and 158 in theN-terminal domain, which contains the ApoE receptor-binding domain.These structural variations are thought to give rise to distinctfunctional attributes among the variants. Consistent with this, thethree ApoE isoforms differ in their binding affinity for members of theLDL receptor family, lipoprotein-binding preferences, and N-terminusstability. Namely, ApoE2 has 50- to 100-fold attenuated LDL receptorbinding ability compared to ApoE3 and ApoE4 (Weisgraber et al., 1982),while ApoE4, unlike the other two variants, preferentially binds tolarge lower-density lipoproteins (Weisgraber et al., 1990) and exhibitsthe lowest N-terminus stability (Morrow et al., 2000). These functionaldifferences confer pathophysiological properties to select ApoEisoforms. While ApoE3, found in 78% of the population, is considered aneutral allele, ApoE2 is associated with type III hyperlipoprotenemia(Hatters et al., 2006) and ApoE4 represents the major known genetic riskfactor for Alzheimer's disease (Corder et al., 1993) and also correlateswith a modest increase in the risk of developing cardiovascular disease(Luc et al., 1994). Given that the multiple human melanoma cell linesanalyzed in the above study are homozygous for the ApoE3 allele, as wellas the ability of recombinant ApoE3 to inhibit melanoma invasion andendothelial recruitment, the above findings are consistent with ApoE3being sufficient and required for the suppression of melanoma metastaticprogression. However, it will be of interest in the future to determinewhether ApoE2 and ApoE4 can modulate these pro-metastatic phenotypes toa similar extent as ApoE3 and whether specific ApoE genotypes conferenhanced risk of melanoma metastatic progression.

Besides surgical resection of primary melanoma lesions, there arecurrently no effective therapies for the prevention of melanomametastasis with interferon therapy increasing overall survival rates at5 years by a meager 3% based on meta-analyses, while phase III trialdata demonstration of significant survival benefits is still outstanding(Garbe et al., 2011). The dramatic enhancement of melanoma metastaticprogression in the context of genetic ablation of systemic ApoE suggeststhat modulating ApoE levels may have significant therapeuticimplications for melanoma—a disease that claims approximately 48,000lives a year globally (Lucas et al., 2006). Given the robust ability ofApoE to suppress melanoma invasion, endothelial migration, metastaticangiogenesis, and metastatic colonization, therapeutic approaches aimedat pharmacological induction of endogenous ApoE levels may significantlyreduce melanoma mortality rates by decreasing metastatic incidence.

The above-described unbiased in vivo selection based approach led todiscovery of deregulated miRNAs that synergistically and dramaticallypromote metastasis by cancer cells from independent patients' melanomacell lines representing both melanotic and amelanotic melanomas. WhilemiR-1908 has not been previously characterized, miR-199a has beenimplicated in hepatocellular carcinoma (Hou et al., 2011; Shen et al.,2010) and osteosarcoma (Duan et al., 2011) that, contrary to melanoma,display down-regulation of miR-199a expression levels. These differencesare consistent with the established tissue-specific expression profilesof miRNAs in various cancer types. The identification of miR-199a as apromoter of melanoma metastasis is supported by a previous clinicalassociation study revealing that increased miR-199a levels correlatewith uveal melanoma progression (Worley et al., 2008), suggesting thatinduced miR-199a expression may be a defining feature of metastaticmelanoma regardless of site of origin. Previous studies have implicatedadditional miRNAs in promoting melanoma metastatic progression such asmiR-182 (Segura et al., 2009), miR-214 (which was upregulated inmetastatic melanoma cells, but it did not functionally perform in theabove studies; Penna et al., 2011), and miR-30b/miR-30d (Gaziel-Sovranet al., 2011). Each of these miRNAs have been reported to only modestlymodulate melanoma metastasis, leading to 1.5- to 2-fold increased ordecreased metastasis modulation upon miRNA over-expression orknock-down, respectively. In contrast, over-expression of eithermiR-199a or miR-1908 enhanced metastasis by 9-fold (FIG. 1C), whilecombinatorial miRNA knock-down synergistically suppressed melanomametastasis by over 70-fold (FIG. 7E). Therefore, the study disclosedherein represents the first systematic discovery of multiple miRNAs thatconvergently and robustly promote human melanoma metastasis, as well asthe first to assign dual cell-autonomous/non-cell-autonomous roles toendogenous metastasis-regulatory miRNAs in cancer.

Previous systematic analysis of miRNAs in breast cancer revealedprimarily a decrease in the expression levels of multiple microRNAs inin vivo selected metastatic breast cancer cells (Tavazoie et al., 2008).Those findings were consistent with the subsequent discovery of manyadditional metastasis suppressor miRNAs in breast cancer (Shi et al.,2010; Wang and Wang, 2011), the identification of a number of miRNAs asdirect transcriptional targets of the p53 tumour suppressor (He et al.,2007), the downregulation of miRNAs in breast cancer relative to normaltissues (Calin and Groce, 2006; Iorio et al., 2005), the downregulationof drosha and dicer in breast cancer (Yan et al., 2011) and metastaticbreast cancer (Grelier et al., 2011), as well as the pro-tumorigenic andpro-metastatic effects of global miRNA silencing through dicerknock-down (Kumar et al., 2007; Kumar et al., 2009; Martello et al.,2010; Noh et al., 2011). In contrast to breast cancer, the abovefindings in melanoma reveal a set of miRNAs upregulated in metastatichuman melanoma, raising the intriguing possibility that miRNA processingmay actually act in a pro-tumorigenic or pro-metastatic manner inmelanoma. Consistent with this, dicer is required for melanocyticdevelopment (Levy et al., 2010), and dicer expression was recently foundto positively correlate with human melanoma progression in aclinico-pathological study (Ma et al., 2011). These findings, whenintegrated with the findings disclosed here, motivate future studies toinvestigate the functional requirement for dicer (Bernstein et al.,2001) in melanoma metastasis.

The establishment of in vivo selection models of melanotic andamelanotic melanoma metastasis has allowed one to identify the cellularphenotypes displayed by highly metastatic melanoma cells. The workreveals that, in addition to enhanced invasiveness, the capacity ofmelanoma cells to recruit endothelial cells is significantly enhanced inhighly metastatic melanoma cells relative to poorly metastatic melanomacells. Additionally, it was found that three major post-transcriptionalregulators of metastasis strongly mediate endothelial recruitment. Itwas further found that the downstream signaling pathway modulated bythese miRNAs also regulates endothelial recruitment. These findingsreveal endothelial recruitment to be a defining feature of metastaticmelanoma cells. Enhanced endothelial recruitment capacity was alsorecently found to be a defining feature of metastatic breast cancer,wherein suppression of metastasis by miR-126 was mediated through miRNAtargeting of two distinct signaling pathways that promote endothelialrecruitment (Png et al., 2012). In breast cancer, endothelialrecruitment increased the likelihood of metastatic initiation ratherthan tumor growth. Similarly, the melanoma metastasis promoter miRNAsstudied here dramatically enhanced metastatic colonization, withoutenhancing primary tumor growth, and increased the number of metastaticnodules—consistent with a role for these miRNAs and their regulatorynetwork in metastatic initiation rather than tumor growth promotion.Taken together, these findings are consistent with endothelialrecruitment into the metastatic niche acting as a promoter of metastaticinitiation and colonization in these distinct epithelial cancer types.Such a non-canonical role for endothelial cells in cancer progressionwould contrast with the established role of endothelial cells inangiogenic enhancement of blood flow spurring enhanced tumor growth.Endothelial cells are known to play such non-canonical roles indevelopment by supplying cues to neighboring cells during organogenesis(Lammert et al., 2001). Such cues have also been recently shown topromote organ regeneration (Ding et al., 2011; Ding et al., 2010;Kobayashi et al., 2010). Future work is needed to determine themetastasis stimulatory factors provided by endothelial cells thatcatalyze metastatic initiation.

The ability of miR-199a-3p, miR-199a-5p, and miR-1908 to individuallypredict metastasis-free survival in a cohort of melanoma patientsindicates the significance of each miRNA as a clinical predictor ofmelanoma cancer progression. Importantly, the dramatic and highlysignificant capacity of the three miRNA aggregate signature (FIG. 7D) tostratify patients at high risk from those at essentially no risk formetastatic relapse reveals both the cooperativity of these miRNAs, aswell as their clinical potential as melanoma biomarkers (Sawyers, 2008)for identifying the subset of patients that might benefit from miRNAinhibition therapy. Therapeutic miRNA targeting has gained momentumthrough the use of in vivo LNAs that have been shown to antagonizemiRNAs in mice (Elmer et al., 2008(b); Krutzfeldt et al., 2005; Obad etal., 2011) and primates (Elmer et al., 2008(a)) and are currently beingtested in human clinical trials. The powerful prognostic capacity of thethree miRNAs, proof-of-principle demonstration of robust synergisticmetastasis prevention achieved by treating highly metastatic melanomacells with a cocktail of LNAs targeting miR-199a-3p, miR-199a-5p, andmiR-1908 (FIG. 7E), as well as the metastasis suppression effect oftherapeutically delivered in vivo-optimized LNAs targeting these miRNAs(FIG. 7J) motivate future clinical studies aimed at determining thetherapeutic potential of combinatorially targeting these pro-metastaticand pro-angiogenic miRNAs in patients at high risk for melanomametastasis—an outcome currently lacking effective chemotherapeuticoptions.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated in their entireties.

1-92. (canceled)
 93. A method of treating metastatic cancer in a subjectin need thereof, the method comprising administering to the subject anLXRβ agonist selected from:

or a pharmaceutically acceptable salt thereof, in an amount sufficientto suppress progression of the metastatic cancer.
 94. The method ofclaim 93, wherein the metastatic cancer is breast cancer, lung cancer,bladder cancer, head and neck cancer, renal cell cancer, colorectalcancer, lymphoma, hepatocellular carcinoma, or prostate cancer.
 95. Themethod of claim 93, wherein the metastatic cancer is breast cancer. 96.The method of claim 95, wherein the breast cancer is triple negativebreast cancer.
 97. The method of claim 93, wherein the metastatic canceris lung cancer.
 98. The method of claim 97, wherein the lung cancer isnon-small cell lung cancer.
 99. The method of claim 97, wherein the lungcancer is small cell lung cancer.
 100. The method of claim 93, whereinthe metastatic cancer is bladder cancer.
 101. The method of claim 93,wherein the metastatic cancer is head and neck cancer.
 102. The methodof claim 93, wherein the metastatic cancer is renal cell cancer. 103.The method of claim 93, wherein the metastatic cancer is colorectalcancer.
 104. The method of claim 93, wherein the metastatic cancer islymphoma.
 105. The method of claim 93, wherein the metastatic cancer ishepatocellular carcinoma.
 106. The method of claim 93, wherein themetastatic cancer is prostate cancer.
 107. The method of claim 93,wherein the LXRβ agonist is compound 1, or a pharmaceutically acceptablesalt thereof.
 108. The method of claim 93, wherein the LXRβ agonist iscompound 2, or a pharmaceutically acceptable salt thereof.
 109. Themethod of claim 93, wherein the LXRβ agonist is compound 12, or apharmaceutically acceptable salt thereof.
 110. The method of claim 93,wherein the LXRβ agonist is compound 25, or a pharmaceuticallyacceptable salt thereof.
 111. The method of claim 93, wherein themetastatic cancer is drug resistant.
 112. The method of claim 111,wherein the metastatic cancer is resistant to platinum-containingchemotherapy, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor,an IDO inhibitor, an anti-hormonal therapy, an antimitotic agent, anangiogenesis inhibitor, and/or a kinase inhibitor.
 113. The method ofclaim 111, wherein the metastatic cancer progressed on or aftertreatment with platinum-containing chemotherapy, a PD-1 inhibitor, aPD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, an anti-hormonaltherapy, an antimitotic agent, an angiogenesis inhibitor, and/or akinase inhibitor.
 114. The method of claim 111, wherein the metastaticcancer has been determined to be, or is predicted to be, resistant toplatinum-containing chemotherapy, a PD-1 inhibitor, a PD-L1 inhibitor, aCTLA-4 inhibitor, an IDO inhibitor, an anti-hormonal therapy, anantimitotic agent, an angiogenesis inhibitor, and/or a kinase inhibitor.115. The method of claim 93, wherein the method does not comprisefurther concurrently administering to the subject an antiproliferativeagent.
 116. The method of claim 93, wherein the method further comprisesadministration of an additional anticancer therapy.
 117. The method ofclaim 116, wherein the additional anticancer therapy is aplatinum-containing chemotherapy, an immunomodulatory, an anti-hormonaltherapy, an antimitotic agent, an angiogenesis inhibitor, and/or akinase inhibitor.
 118. The method of claim 117, wherein theplatinum-containing chemotherapy is oxaliplatin, carboplatin, orcisplatin, the immunomodulator is a PD-1 inhibitor, a CTLA4 inhibitor,an IDO inhibitor, and/or a PDL1 inhibitor, the kinase inhibitor iserlotinib or trastuzumab, the angiogenesis inhibitor is bevacizumab,and/or the antimitotic agent is paclitaxel or docetaxel.
 119. The methodof claim 93, wherein the method comprises reducing or stopping theformation of new metastatic tumors and/or reducing, stopping, orreversing the load of a metastatic tumor.
 120. The method of claim 93,wherein the method comprises the suppression of metastatic colonizationof the metastatic cancer.
 121. The method of claim 120, wherein themethod comprises the suppression of metastatic colonization of themetastatic cancer to the lung and/or the brain.